Publications by authors named "Thierry Pourcher"

34 Publications

Combined Omic Analyzes of Cerebral Thrombi: A New Molecular Approach to Identify Cardioembolic Stroke Origin.

Stroke 2021 May 21:STROKEAHA120032129. Epub 2021 May 21.

Laboratory Transporter in Imaging and Radiotherapy in Oncology (TIRO), Direction de la Recherche Fondamentale (DRF), Institut des sciences du vivant Fréderic Joliot, Commissariat à l'Energie Atomique et aux énergies alternatives (CEA), Université Côte d'Azur (UCA), Nice, France (L.S., J.-M.G., F.G., S.L., T.P.).

Background And Purpose: The diagnosis of cardioembolic stroke can be challenging for patient management in secondary stroke prevention, particularly in the case of covert paroxysmal atrial fibrillation. The molecular composition of a cerebral thrombus is related to its origin. Therefore, proteomic and metabolomic analyses of the retrieved thrombotic material should allow the identification of biomarkers or signatures to improve the etiological diagnosis of stroke.

Methods: In this pilot study, the proteome and metabolome of cerebral thrombi from atherothrombotic and cardioembolic stroke patients were studied according to ASCOD phenotyping (A: atherosclerosis; S: small-vessel disease; C: cardiac pathology; O: other causes; D: dissection), with the highest causality grade, from the ThrombiOMIC cohort (consecutive patients with stroke recanalized by mechanical thrombectomy in an acute phase). Proteomic and metabolomic results were used separately or combined, and the obtained omic signatures were compared with classical cardioembolic stroke predictors using pairwise comparisons of the area under receiver operating characteristics.

Results: Among 59 patients of the ThrombiOMIC cohort, 34 patients with stroke showed a cardioembolic phenotype and 7 had an atherothrombotic phenotype. Two thousand four hundred fifty-six proteins and 5019 molecular features of the cerebral thrombi were identified using untargeted proteomic and metabolomic approaches, respectively. Area under receiver operating characteristics to predict the cardioembolic origin of stroke were calculated using the proteomic results (0.945 [95% CI, 0.871-1]), the metabolomic results (0.836 [95% CI, 0.714-0.958]), and combined signatures (0.996 [95% CI, 0.984-1]). The diagnostic performance of the combined signatures was significantly higher than that of classical predictors such as the plasmatic BNP (B-type natriuretic peptide) level (area under receiver operating characteristics, 0.803 [95% CI, 0.629-0.976]).

Conclusions: The combined proteomic and metabolomic analyses of retrieved cerebral thrombi is a very promising molecular approach to predict the cardioembolic cause of stroke and to improve secondary stroke prevention strategies.
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http://dx.doi.org/10.1161/STROKEAHA.120.032129DOI Listing
May 2021

Quantitative phase microscopy for non-invasive live cell population monitoring.

Sci Rep 2021 Feb 24;11(1):4409. Epub 2021 Feb 24.

Transporter in Imaging and Radiotherapy in Oncology (TIRO), Institut des Sciences et Biotechnologies du Vivant Frédéric Joliot, CEA, School of Medicine, 28 Av de Valombrose, 06107, Nice, France.

We present here a label-free development based on preexisting Quantitative Phase Imaging (QPI) that allows non-invasive live monitoring of both individual cells and cell populations. Growth, death, effect of toxic compounds are quantified under visible light with a standard inverted microscope. We show that considering the global biomass of a cell population is a more robust and accurate method to assess its growth parameters in comparison to compiling individually segmented cells. This is especially true for confluent conditions. This method expands the use of light microscopy in answering biological questions concerning live cell populations even at high density. In contrast to labeling or lysis of cells this method does not alter the cells and could be useful in high-throughput screening and toxicity studies.
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http://dx.doi.org/10.1038/s41598-021-83537-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904828PMC
February 2021

Ingested Ketone Ester Leads to a Rapid Rise of Acetyl-CoA and Competes with Glucose Metabolism in the Brain of Non-Fasted Mice.

Int J Mol Sci 2021 Jan 7;22(2). Epub 2021 Jan 7.

Laboratory Transporter in Imaging and Radiotherapy in Oncology (TIRO), Direction de la Recherche Fondamentale (DRF), Institut des Sciences du Vivant Fréderic Joliot, Commissariat à l'Energie Atomique et aux Énergies Alternatives (CEA), University Côte d'Azur, F-06107 Nice, France.

The role of ketone bodies in the cerebral energy homeostasis of neurological diseases has begun to attract recent attention particularly in acute neurological diseases. In ketogenic therapies, ketosis is achieved by either a ketogenic diet or by the administration of exogenous ketone bodies. The oral ingestion of the ketone ester (KE), (R)-3-hydroxybutyl (R)-3-hydroxybutyrate, is a new method to generate rapid and significant ketosis (i.e., above 6 mmol/L) in humans. KE is hydrolyzed into β-hydroxybutyrate (βHB) and its precursor 1,3-butanediol. Here, we investigate the effect of oral KE administration (3 mg KE/g of body weight) on brain metabolism of non-fasted mice using liquid chromatography in tandem with mass spectrometry. Ketosis (Cmax = 6.83 ± 0.19 mmol/L) was obtained at Tmax = 30 min after oral KE-gavage. We found that βHB uptake into the brain strongly correlated with the plasma βHB concentration and was preferentially distributed in the neocortex. We showed for the first time that oral KE led to an increase of acetyl-CoA and citric cycle intermediates in the brain of non-fasted mice. Furthermore, we found that the increased level of acetyl-CoA inhibited glycolysis by a feedback mechanism and thus competed with glucose under physiological conditions. The brain pharmacodynamics of this oral KE strongly suggest that this agent should be considered for acute neurological diseases.
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http://dx.doi.org/10.3390/ijms22020524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825708PMC
January 2021

Metabolome of Cerebral Thrombi Reveals an Association between High Glycemia at Stroke Onset and Good Clinical Outcome.

Metabolites 2020 Nov 25;10(12). Epub 2020 Nov 25.

Laboratory Transporter in Imaging and Radiotherapy in Oncology (TIRO), Direction de la Recherche Fondamentale (DRF), Institut des sciences du vivant Fréderic Joliot, Commissariat à l'Energie Atomique et aux énergies alternatives (CEA), University Côte d'Azur, F-06107 Nice, France.

Despite the fact that glucose is the main fuel of the brain, hyperglycemia at hospital admission is generally associated with a poor functional outcome in stroke patients. This paradox may be explained by the lack of information about the blood glucose level at stroke onset. Here, we analyzed the metabolome of blood cells entrapped in cerebral thrombi to gain insight into their metabolism at stroke onset. Fourty-one consecutive stroke patients completely recanalized by mechanical thrombectomy within 6 h were included. The metabolome of retrieved thrombi was analyzed by liquid chromatography tandem with mass spectrometry. Discriminant Analysis (sparse Partial Least Squares Discriminant Analysis (sPLS-DA)) was performed to identify classification models and significant associated features of favorable clinical outcome at 3 months (modified Rankin Scale (mRS) < 2). sPLS-DA of the metabolomes of cerebral thrombi discriminated between stroke patients with a favorable or poor clinical outcome (Area Under the Curve (AUC) = 0.992 (0.931-1)). In addition, our results revealed that high sorbitol and glucose levels in the thrombi positively correlated with favorable clinical outcomes. Sorbitol, a short-term glycemic index reflecting a high blood glucose level at stroke onset, was found to be an independent predictor of good outcome (AUC = 0.908 (0.807-0.995)). This study demonstrates that a high blood glucose level at stroke onset is beneficial to the clinical outcome of the patient.
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http://dx.doi.org/10.3390/metabo10120483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760729PMC
November 2020

Tumor microenvironment affects exogenous sodium/iodide symporter expression.

Transl Oncol 2021 Jan 17;14(1):100937. Epub 2020 Nov 17.

Transporters in Imaging and Radiotherapy in Oncology (TIRO), School of Medicine, Direction de la Recherche Fondamentale (DRF), Institut des sciences du vivant Fréderic Joliot, Commissariat à l'Energie Atomique et aux énergies alternatives (CEA), Université Côte d'Azur (UCA), 28 Avenue de Valombrose, 06107 Nice, France. Electronic address:

For decades, sodium/iodide symporter NIS-mediated iodide uptake has played a crucial role in the radioactive ablation of thyroid cancer cells. NIS-based gene therapy has also become a promising tool for the treatment of tumors of extrathyroidal origin. But its applicability has been hampered by reduced expression of NIS, resulting in a moderated capacity to accumulate I and in inefficient ablation. Despite numerous preclinical enhancement strategies, the understanding of NIS expression within tumors remains limited. This study aims at a better understanding of the functional behavior of exogenous NIS expression in the context of malignant solid tumors that are characterized by rapid growth with an insufficient vasculature, leading to hypoxia and quiescence. Using subcutaneous HT29NIS and K7M2NIS tumors, we show that NIS-mediated uptake and NIS expression at the plasma membrane of cancer cells are impaired in the intratumoral regions. For a better understanding of the underlying molecular mechanisms induced by hypoxia and quiescence (separately and in combination), we performed experiments on HT29NIS cancer cells. Hypoxia and quiescence were both found to impair NIS-mediated uptake through mechanisms including NIS mis-localization. Modifications in the expression of proteins and metabolites involved in plasma membrane localization and in energy metabolism were found using untargeted proteomics and metabolomics approaches. In conclusion, our results provide evidence that hypoxia and quiescence impair NIS expression at the plasma membrane, and iodide uptake. Our study also shows that the tumor microenvironment is an important parameter for successful NIS-based cancer treatment.
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http://dx.doi.org/10.1016/j.tranon.2020.100937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679261PMC
January 2021

Comparison of unsupervised machine-learning methods to identify metabolomic signatures in patients with localized breast cancer.

Comput Struct Biotechnol J 2020 3;18:1509-1524. Epub 2020 Jun 3.

University Côte d'Azur, Epidemiology and Biostatistics Department, Centre Antoine Lacassagne, Nice F-06189, France.

Genomics and transcriptomics have led to the widely-used molecular classification of breast cancer (BC). However, heterogeneous biological behaviors persist within breast cancer subtypes. Metabolomics is a rapidly-expanding field of study dedicated to cellular metabolisms affected by the environment. The aim of this study was to compare metabolomic signatures of BC obtained by 5 different unsupervised machine learning (ML) methods. Fifty-two consecutive patients with BC with an indication for adjuvant chemotherapy between 2013 and 2016 were retrospectively included. We performed metabolomic profiling of tumor resection samples using liquid chromatography-mass spectrometry. Here, four hundred and forty-nine identified metabolites were selected for further analysis. Clusters obtained using 5 unsupervised ML methods (PCA k-means, sparse k-means, spectral clustering, SIMLR and k-sparse) were compared in terms of clinical and biological characteristics. With an optimal partitioning parameter k = 3, the five methods identified three prognosis groups of patients (favorable, intermediate, unfavorable) with different clinical and biological profiles. SIMLR and K-sparse methods were the most effective techniques in terms of clustering. survival analysis revealed a significant difference for 5-year predicted OS between the 3 clusters. Further pathway analysis using the 449 selected metabolites showed significant differences in amino acid and glucose metabolism between BC histologic subtypes. Our results provide proof-of-concept for the use of unsupervised ML metabolomics enabling stratification and personalized management of BC patients. The design of novel computational methods incorporating ML and bioinformatics techniques should make available tools particularly suited to improving the outcome of cancer treatment and reducing cancer-related mortalities.
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http://dx.doi.org/10.1016/j.csbj.2020.05.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327012PMC
June 2020

Proteomic Analysis of Iodinated Contrast Agent-Induced Perturbation of Thyroid Iodide Uptake.

J Clin Med 2020 Jan 23;9(2). Epub 2020 Jan 23.

Université Côte d'Azur, UMR E4320, CEA, F-06107 Nice, France.

(1) Background: We recently showed that iodinated contrast media (ICM) reduced thyroid uptake of iodide independently of free iodide through a mechanism different from that of NaI and involving a dramatic and long-lasting decrease in Na/I symporter expression. The present study aimed at comparing the response of the thyroid to ICM and NaI using a quantitative proteomic approach. (2) Methods: Scintiscans were performed on ICM-treated patients. Micro Single-Photon Emission Computed Tomography (microSPECT/CT) imaging was used to assess thyroid uptakes in ICM- or NaI-treated mice and their response to recombinant human thyroid-stimulating hormone. Total thyroid iodide content and proteome was determined in control, NaI-, or ICM-treated animals. (3) Results: The inhibitory effect of ICM in patients was selectively observed on thyroids but not on salivary glands for up to two months after a systemic administration. An elevated level of iodide was observed in thyroids from NaI-treated mice but not in those from ICM animals. Exposure of the thyroid to NaI modulates 15 cellular pathways, most of which are also affected by ICM treatment (including the elF4 and P706SK cell signaling pathway and INSR identified as an upstream activator in both treatments). In addition, ICM modulates 16 distinct pathways and failed to affect thyroid iodide content. Finally, administration of ICM reduces thyroid-stimulating hormone (TSH) receptor expression which results in a loss of TSH-induced iodide uptake by the thyroid. (4) Conclusions: Common intracellular mechanisms are involved in the ICM- and NaI-induced reduction of iodide uptake. However, ICM fails to affect thyroid iodide content which suggests that the modulation of these common pathways is triggered by separate effectors. ICM also modulates numerous distinct pathways which may account for its long-lasting effect on thyroid uptake. These observations may have implications in the management of patients affected by differentiated thyroid carcinomas who have been exposed to ICM. They also provide the basis for the utilization of ICM-based compounds in radioprotection of the thyroid.
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http://dx.doi.org/10.3390/jcm9020329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073936PMC
January 2020

Improving I Radioiodine Therapy By Hybrid Polymer-Grafted Gold Nanoparticles.

Int J Nanomedicine 2019 30;14:7933-7946. Epub 2019 Sep 30.

Laboratory Transporter in Imaging and Radiotherapy in Oncology (TIRO), Institut de Biosciences et Biotechnologies d'Aix-Marseille (BIAM), Commissariat à l'Energie Atomique, Nice, France.

Background: Human trials combining external radiotherapy (RT) and metallic nanoparticles are currently underway in cancer patients. For internal RT, in which a radioisotope such as radioiodine is systemically administered into patients, there is also a need for enhancing treatment efficacy, decreasing radiation-induced side effects and overcoming radio-resistance. However, if strategies vectorising radioiodine through nanocarriers have been documented, sensitizing the neoplasm through the use of nanotherapeutics easily translatable to the clinic in combination with the standard systemic radioiodine treatment has not been assessed yet.

Method And Materials: The present study explored the potential of hybrid poly(methacrylic acid)-grafted gold nanoparticles to improve the performances of systemic I-mediated RT on cancer cells and in tumor-bearing mice. Such nanoparticles were chosen based on their ability previously described by our group to safely withstand irradiation doses while exhibiting good biocompatibility and enhanced cellular uptake.

Results: In vitro clonogenic assays performed on melanoma and colorectal cancer cells showed that poly(methacrylic acid)-grafted gold nanoparticles (PMAA-AuNPs) could efficiently lead to a marked tumor cell mortality when combined to a low activity of radioiodine, which alone appeared to be essentially ineffective on tumor cells. In vivo, tumor enrichment with PMAA-AuNPs significantly enhanced the killing potential of a systemic radioiodine treatment.

Conclusion: This is the first report of a simple and reliable nanomedicine-based approach to reduce the dose of radioiodine required to reach curability. In addition, these results open up novel perspectives for using high-Z metallic NPs in additional molecular radiation therapy demonstrating heterogeneous dose distributions.
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http://dx.doi.org/10.2147/IJN.S211496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6777639PMC
January 2020

LC-MS based metabolomic profiling for renal cell carcinoma histologic subtypes.

Sci Rep 2019 Oct 30;9(1):15635. Epub 2019 Oct 30.

Central Laboratory of Anatomopathology, Centre Hospitalier Universitaire, Nice, France.

Renal cell carcinomas (RCC) are classified according to their histological features. Accurate classification of RCC and comprehensive understanding of their metabolic dysregulation are of critical importance. Here we investigate the use of metabolomic analyses to classify the main RCC subtypes and to describe the metabolic variation for each subtype. To this end, we performed metabolomic profiling of 65 RCC frozen samples (40 clear cell, 14 papillary and 11 chromophobe) using liquid chromatography-mass spectrometry. OPLS-DA multivariate analysis based on metabolomic data showed clear discrimination of all three main subtypes of RCC (R = 75.0%, Q = 59.7%). The prognostic performance was evaluated using an independent cohort and showed an AUROC of 0.924, 0.991 and 1 for clear cell, papillary and chromophobe RCC, respectively. Further pathway analysis using the 21 top metabolites showed significant differences in amino acid and fatty acid metabolism between three RCC subtypes. In conclusion, this study shows that metabolomic profiling could serve as a tool that is complementary to histology for RCC subtype classification. An overview of metabolic dysregulation in RCC subtypes was established giving new insights into the understanding of their clinical behaviour and for the development of targeted therapeutic strategies.
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http://dx.doi.org/10.1038/s41598-019-52059-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821699PMC
October 2019

Urinary ketone body loss leads to degeneration of brain white matter in elderly SLC5A8-deficient mice.

J Cereb Blood Flow Metab 2020 08 10;40(8):1709-1723. Epub 2019 Sep 10.

Laboratory Transporter in Imaging and Radiotherapy in Oncology (TIRO), University Nice Sophia Antipolis, Institut de biosciences et biotechnologies d'Aix-Marseille (BIAM), Commissariat a l'Energie Atomique, University Côte d'Azur, Nice, France.

SLC5A8 is a sodium-coupled monocarboxylate and ketone transporter expressed in various epithelial cells. A putative role of SLC5A8 in neuroenergetics has been also hypothesized. To clarify this issue, we studied the cerebral phenotype of SLC5A8-deficient mice during aging. Elderly SLC5A8-deficient mice presented diffuse leukoencephalopathy characterized by intramyelinic oedema without demyelination suggesting chronic energetic crisis. Hypo-metabolism in the white matter of elderly SLC5A8-deficient mice was found using Tc-hexamethylpropyleneamine oxime (HMPAO) single-photon emission CT (SPECT). Since the SLC5A8 protein could not be detected in the mouse brain, it was hypothesized that the leukoencephalopathy of aging SLC5A8-deficient mice was caused by the absence of slc5a8 expression in a peripheral organ, i.e. the kidney, where SLC5A8 is strongly expressed. A hyper-excretion of the ketone β-hydroxybutyrate (BHB) in the urine of SLC5A8-deficient mice was observed and showed that SLC5A8-deficient mice suffered a cerebral BHB insufficiency. Elderly SLC5A8-deficient mice also presented altered glucose metabolism. We propose that the continuous renal loss of BHB leads to a chronic energetic deficiency in the brain of elderly SLC5A8-deficient mice who are unable to counterbalance their glucose deficit. This study highlights the importance of alternative energetic substrates in neuroenergetics especially under conditions of restricted glucose availability.
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http://dx.doi.org/10.1177/0271678X19873662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370371PMC
August 2020

RGD-functionalized magnetosomes are efficient tumor radioenhancers for X-rays and protons.

Nanomedicine 2020 01 24;23:102084. Epub 2019 Aug 24.

Laboratoire TIRO, UMRE 4320, BIAM, DRT, CEA, Nice Côte d'Azur University, France; Federation Claude Lalanne, Nice Côte d'Azur University, France. Electronic address:

Although chemically synthesized ferro/ferrimagnetic nanoparticles have attracted great attention in cancer theranostics, they lack radio-enhancement efficacy due to low targeting and internalization ability. Herein, we investigated the potential of RGD-tagged magnetosomes, bacterial biogenic magnetic nanoparticles naturally coated with a biological membrane and genetically engineered to express an RGD peptide, as tumor radioenhancers for conventional radiotherapy and proton therapy. Although native and RGD-magnetosomes similarly enhanced radiation-induced damage to plasmid DNA, RGD-magnetoprobes were able to boost the efficacy of radiotherapy to a much larger extent than native magnetosomes both on cancer cells and in tumors. Combined to [email protected], proton therapy exceeded the efficacy of X-rays at equivalent doses. Also, increased secondary emissions were measured after irradiation of magnetosomes with protons versus photons. Our results indicate the therapeutic advantage of using functionalized magnetoparticles to sensitize tumors to both X-rays and protons and strengthen the case for developing biogenic magnetoparticles for multimodal nanomedicine in cancer therapy.
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http://dx.doi.org/10.1016/j.nano.2019.102084DOI Listing
January 2020

A Carboxy-Terminal Monoleucine-Based Motif Participates in the Basolateral Targeting of the Na+/I- Symporter.

Endocrinology 2019 01;160(1):156-168

Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

The Na+/iodide (I-) symporter (NIS), a glycoprotein expressed at the basolateral plasma membrane of thyroid follicular cells, mediates I- accumulation for thyroid hormonogenesis and radioiodide therapy for differentiated thyroid carcinoma. However, differentiated thyroid tumors often exhibit lower I- transport than normal thyroid tissue (or even undetectable I- transport). Paradoxically, the majority of differentiated thyroid cancers show intracellular NIS expression, suggesting abnormal targeting to the plasma membrane. Therefore, a thorough understanding of the mechanisms that regulate NIS plasma membrane transport would have multiple implications for radioiodide therapy. In this study, we show that the intracellularly facing carboxy-terminus of NIS is required for the transport of the protein to the plasma membrane. Moreover, the carboxy-terminus contains dominant basolateral information. Using internal deletions and site-directed mutagenesis at the carboxy-terminus, we identified a highly conserved monoleucine-based sorting motif that determines NIS basolateral expression. Furthermore, in clathrin adaptor protein (AP)-1B-deficient cells, NIS sorting to the basolateral plasma membrane is compromised, causing the protein to also be expressed at the apical plasma membrane. Computer simulations suggest that the AP-1B subunit σ1 recognizes the monoleucine-based sorting motif in NIS carboxy-terminus. Although the mechanisms by which NIS is intracellularly retained in thyroid cancer remain elusive, our findings may open up avenues for identifying molecular targets that can be used to treat radioiodide-refractory thyroid tumors that express NIS intracellularly.
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http://dx.doi.org/10.1210/en.2018-00603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936561PMC
January 2019

DO MULTIPLE ADMINISTRATIONS OF STABLE IODINE PROTECT POPULATION CHRONICALLY EXPOSED TO RADIOACTIVE IODINE: WHAT IS PRIODAC RESEARCH PROGRAM (2014-22) TEACHING US?

Radiat Prot Dosimetry 2018 Dec;182(1):67-79

Institut de Radioprotection et de Sûreté Nucléaire (IRSN), PSE-Santé, Fontenay-aux-Roses, France.

Single dose of potassium iodide (KI) is recommended to prevent the risk of thyroid cancer during nuclear accidents. However in the case of repeated/protracted radioiodine release, a unique dose of KI may not protect efficiently the thyroid against the risk of further developing a radiation-induced cancer. The new WHO guidelines for the use in planning for and responding to radiological and nuclear emergencies identify the need of more data on this subject as one of the four research priorities. The aims of the PRIODAC project are (1) to assess the associated side effects of repeated intakes of KI, (2) to better understand the molecular mechanisms regulating the metabolism of iodine, (3) to revise the regulatory French marketing authorization of 65-mg KI tablets and (4) to develop new recommendations related to the administration of KI toward a better international harmonization. A review of the literature and the preliminary data are presented here.
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http://dx.doi.org/10.1093/rpd/ncy129DOI Listing
December 2018

Iodinated Contrast Agents Perturb Iodide Uptake by the Thyroid Independently of Free Iodide.

J Nucl Med 2018 01 19;59(1):121-126. Epub 2017 Oct 19.

Laboratoire TIRO, UMRE 4320, CEA, Nice, France.

Perturbation of thyroid iodide uptake is a well-documented side effect of the use of iodinated contrast media (ICM) administered intravenously. This side effect is thought to be mediated by free iodide in ICM formulations, but this hypothesis has never been formally proven. The aim of the present study was to assess the validity of this hypothesis. We used mass spectrometry analysis to quantify free-iodide contamination in ICM. Established cell lines expressing the Na/I symporter (NIS) were used to quantify the effect of ICM on iodide uptake. SPECT/CT was used to measure the in vivo uptake of Tc-pertechnetate and I in 2 NIS-expressing mouse tissues, thyroid and salivary glands. Scintiscans of ICM-naïve and ICM-administered patients were compared. Immunohistologic and Western blot analyses were performed to evaluate NIS protein expression in these organs. Although free iodide was present in ICM formulations, in vitro uptake of iodide by NIS-expressing cells was not significantly affected by ICM. In mice, intravenous or sublingual administration of ICM led to a reduction in radiotracer uptake by the thyroid, accompanied by a dramatic reduction in NIS protein expression in this tissue. In the salivary glands, neither radiotracer uptake nor NIS protein expression was affected by ICM. The thyroid-selective effect of ICM was also observed in humans. Administration of potassium iodide as a source of free iodide led to a diminution of Tc-pertechnetate uptake in both mouse thyroid and mouse salivary glands. Altogether, these data rule out a direct intervention of free iodide in the perturbation of thyroid uptake and suggest a direct and selective effect of ICM on the thyroid. We demonstrated that ICM reduce thyroid uptake of iodide independently of free iodide. This effect is due to a specific and dramatic decrease in NIS expression in thyrocytes. These data cast serious doubt on the relevance of measuring urinary iodide concentration to evaluate the delay between ICM administration and radioiodine therapy in patients with differentiated thyroid carcinoma. Finally, the ability of ICM to perturb iodide uptake in the thyroid may be used in radioprotection.
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http://dx.doi.org/10.2967/jnumed.117.195685DOI Listing
January 2018

Single-Photon Emission Computed Tomography for Preclinical Assessment of Thyroid Radioiodide Uptake Following Various Combinations of Preparative Measures.

Thyroid 2016 11 11;26(11):1614-1622. Epub 2016 Aug 11.

1 Laboratory of Transporters, Imaging, Radiotherapy in Oncology, Unité Mixte de Recherche E4320, Institut de Biosciences et Biotechnologies , CEA, Nice, France .

Background: MicroSPECT/CT imaging was used to quantitatively evaluate how iodide uptake in the mouse thyroid is influenced by (i) route of iodine administration; (ii) injection of recombinant human thyrotropin (rhTSH); and (iii) low iodide diet (LID) in euthyroid and triiodothyronine (T3)-treated mice.

Methods: Pertechnetate (TcO) and I thyroid uptake in euthyroid and T3-treated animals fed either a normal-iodine diet (NID) or an LID, treated or not with rhTSH, and radiotracer administered intravenously, subcutaneously, intraperitoneally or by gavage, were assessed using microSPECT/CT imaging. Western blotting was performed to measure sodium/iodide symporter expression levels in the thyroid.

Results: Systemic administration of radioiodide resulted in a higher (2.35-fold in NID mice) accumulation of iodide in the thyroid than oral administration. Mice fed LID with systemic radioiodide administration showed a further two-fold increase in thyroid iodide uptake to yield a ∼5-fold increase in uptake compared to the standard NID/oral route. Although rhTSH injections stimulated thyroid activity in both euthyroid and T3-treated mice fed the NID, uptake levels for T3-treated mice remained low compared with those for the euthyroid mice. Combining LID and rhTSH in T3-treated mice resulted in a 2.8-fold higher uptake compared with NID/T3/rhTSH mice and helped restore thyroid activity to levels equivalent to those of euthyroid animals.

Conclusions: Systemic radioiodide administration results in higher thyroidal iodide levels than oral administration, particularly in LID-fed mice. These data highlight the importance of LID, both in euthyroid and T3-treated, rhTSH-injected mice. Extrapolated to human patients, and in the context of clinical guidelines for the preparation of differentiated thyroid cancer patients, our data indicate that LID can potentiate the efficacy of rhTSH treatment in T3-treated patients.
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http://dx.doi.org/10.1089/thy.2015.0652DOI Listing
November 2016

High-Frequency Quantitative Ultrasound Spectroscopy of Excised Canine Livers and Mouse Tumors Using the Structure Factor Model.

IEEE Trans Ultrason Ferroelectr Freq Control 2016 09 4;63(9):1335-1350. Epub 2016 May 4.

Three scattering models were examined for characterizing ex vivo canine livers and HT29 mouse tumors in the 10-38- and the 15-42-MHz frequency bandwidth, respectively. The spherical Gaussian model (SGM) and the fluid sphere model (FSM) that were examined are suitable for dealing with sparse media, whereas the structure factor model (SFM) is adapted for characterizing concentrated media. For the canine livers, the scatterer radius and the acoustic concentration estimated with the three models were similar and matched well the nuclear structures obtained from histological analysis (with relative errors less than 7%). These results show that the livers could be considered as a diluted medium and that the nuclei in liver could be a dominant source of scattering. For the homogeneous mouse tumors, containing mostly viable HT29 cells, scatterer radius and volume fraction estimated with the SFM showed good agreement with the whole cell structures obtained from histological analysis (with relative errors less than 15%), whereas the sparse models (the SGM and the FSM) gave no consistent quantitative ultrasound parameters. This suggests that the viable HT29 cell areas have densely packed cellular content and that the whole HT29 cell could be responsible for scattering. For the heterogeneous tumors, the hyperechogenic zones observed in the B-mode images were linked to the presence of small necrotic areas surrounded by viable HT29 cells. Comparison between sparse and concentrated models shows that these hyperechogenic zones could be considered as a concentrated medium.
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http://dx.doi.org/10.1109/TUFFC.2016.2563169DOI Listing
September 2016

A systematic evaluation of sorting motifs in the sodium-iodide symporter (NIS).

Biochem J 2016 Apr 1;473(7):919-28. Epub 2016 Feb 1.

Laboratoire TIRO, CEA, Faculté de médecine, 28 Avenue de Valombrose, 06107 Nice, France Laboratoire TIRO, Université de Nice Sophia Antipolis, Faculté de médecine, 28 Avenue de Valombrose, 06107 Nice, France.

The sodium-iodide symporter (NIS) is an integral membrane protein that plays a crucial role in iodide accumulation, especially in the thyroid. As for many other membrane proteins, its intracellular sorting and distribution have a tremendous effect on its function, and constitute an important aspect of its regulation. Many short sequences have been shown to contribute to protein trafficking along the sorting or endocytic pathways. Using bioinformatics tools, we identified such potential sites on human NIS [tyrosine-based motifs, SH2-(Src homology 2), SH3- and PDZ (post-synaptic density-95/discs large tumour suppressor/zonula occludens-1)-binding motifs, and diacidic, dibasic and dileucine motifs] and analysed their roles using mutagenesis. We found that several of these sites play a role in protein stability and/or targeting to the membrane. Aside from the mutation at position 178 (SH2 plus tyrosine-based motif) that affects iodide uptake, the most drastic effect is associated with the mutation of an internal PDZ-binding motif at position 121 that completely abolishes NIS expression at the plasma membrane. Mutating the sites located on the C-terminal domain of the protein has no effect except for the creation of a diacidic motif that decreases the total NIS protein level without affecting its expression at the plasma membrane.
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http://dx.doi.org/10.1042/BJ20151086DOI Listing
April 2016

Evaluation of tetrafunctional block copolymers as synthetic vectors for lung gene transfer.

Biomaterials 2015 Mar 13;45:10-7. Epub 2015 Jan 13.

Laboratoire TIRO, UMRE 4320, iBEB, DSV, CEA, Nice, France; Université de Nice-Sophia Antipolis, Nice, France. Electronic address:

In the present study, we evaluated, in mice, the efficacy of the tetrafunctional block copolymer 704 as a nonviral gene delivery vector to the lungs. SPECT/CT molecular imaging of gene expression, biochemical assays, and immunohistochemistry were used. Our dataset shows that the formulation 704 resulted in higher levels of reporter gene expression than the GL67A formulation currently being used in a clinical trial in cystic fibrosis patients. The inflammatory response associated with this gene transfer was lower than that induced by the GL67A formulation, and the 704 formulation was amenable to repeated administrations. The cell types transfected by the 704 formulation were type I and type II pneumocytes, and transgene expression could not be detected in macrophages. These results emphasize the relevance of the 704 formulation as a nonviral gene delivery vector for lung gene therapy. Further studies will be required to validate this vector in larger animals, in which the lungs are more similar to human lungs.
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http://dx.doi.org/10.1016/j.biomaterials.2014.12.051DOI Listing
March 2015

⁹⁹mTcO₄--, auger-mediated thyroid stunning: dosimetric requirements and associated molecular events.

PLoS One 2014 24;9(3):e92729. Epub 2014 Mar 24.

Laboratoire TIRO, UMRE 4320, iBEB, DSV, CEA, Nice, France; Université de Nice-Sophia Antipolis, Nice, France; Centre Antoine Lacassagne, Department of nuclear medicine, Nice, France.

Low-energy Auger and conversion electrons deposit their energy in a very small volume (a few nm3) around the site of emission. From a radiotoxicological point of view the effects of low-energy electrons on normal tissues are largely unknown, understudied, and generally assumed to be negligible. In this context, the discovery that the low-energy electron emitter, 99mTc, can induce stunning on primary thyrocytes in vitro, at low absorbed doses, is intriguing. Extrapolated in vivo, this observation suggests that a radioisotope as commonly used in nuclear medicine as 99mTc may significantly influence thyroid physiology. The aims of this study were to determine whether 99mTc pertechnetate (99mTcO4-) is capable of inducing thyroid stunning in vivo, to evaluate the absorbed dose of 99mTcO4- required to induce this stunning, and to analyze the biological events associated/concomitant with this effect. Our results show that 99mTcO4--mediated thyroid stunning can be observed in vivo in mouse thyroid. The threshold of the absorbed dose in the thyroid required to obtain a significant stunning effect is in the range of 20 Gy. This effect is associated with a reduced level of functional Na/I symporter (NIS) protein, with no significant cell death. It is reversible within a few days. At the cellular and molecular levels, a decrease in NIS mRNA, the generation of double-strand DNA breaks, and the activation of the p53 pathway are observed. Low-energy electrons emitted by 99mTc can, therefore, induce thyroid stunning in vivo in mice, if it is exposed to an absorbed dose of at least 20 Gy, a level unlikely to be encountered in clinical practice. Nevertheless this report presents an unexpected effect of low-energy electrons on a normal tissue in vivo, and provides a unique experimental setup to understand the fine molecular mechanisms involved in their biological effects.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092729PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3963936PMC
January 2015

The sodium/iodide symporter: state of the art of its molecular characterization.

Biochim Biophys Acta 2014 Jan 27;1838(1 Pt B):244-53. Epub 2013 Aug 27.

SBTN, bât 170, centre de Marcoule, BP 17171, 30207 Bagnols sur Cèze CEDEX, France; Laboratoire TIRO, Faculté de médecine, Université de Nice Sophia-Antipolis, 28 Avenue de Valombrose, 06107 Nice CEDEX, France; CAL, TIRO, F-06107 Nice, France. Electronic address:

The sodium/iodide symporter (NIS or SLC5A5) is an intrinsic membrane protein implicated in iodide uptake into thyroid follicular cells. It plays a crucial role in iodine metabolism and thyroid regulation and its function is widely exploited in the diagnosis and treatment of benign and malignant thyroid diseases. A great effort is currently being made to develop a NIS-based gene therapy also allowing the radiotreatment of nonthyroidal tumors. NIS is also expressed in other tissues, such as salivary gland, stomach and mammary gland during lactation, where its physiological role remains unclear. The molecular identity of the thyroid iodide transporter was elucidated approximately fifteen years ago. It belongs to the superfamily of sodium/solute symporters, SSS (and to the human transporter family, SLC5), and is composed of 13 transmembrane helices and 643 amino acid residues in humans. Knowledge concerning NIS structure/function relationship has been obtained by taking advantage of the high resolution structure of one member of the SSS family, the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT), and from studies of gene mutations leading to congenital iodine transport defects (ITD). This review will summarize current knowledge regarding the molecular characterization of NIS.
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http://dx.doi.org/10.1016/j.bbamem.2013.08.013DOI Listing
January 2014

Normalisation to blood activity is required for the accurate quantification of Na/I symporter ectopic expression by SPECT/CT in individual subjects.

PLoS One 2012 21;7(3):e34086. Epub 2012 Mar 21.

INSERM U948, Biothérapies Hépatiques, CHU Hôtel Dieu, Nantes, France.

The utilisation of the Na/I symporter (NIS) and associated radiotracers as a reporter system for imaging gene expression is now reaching the clinical setting in cancer gene therapy applications. However, a formal assessment of the methodology in terms of normalisation of the data still remains to be performed, particularly in the context of the assessment of activities in individual subjects in longitudinal studies. In this context, we administered to mice a recombinant, replication-incompetent adenovirus encoding rat NIS, or a human colorectal carcinoma cell line (HT29) encoding mouse NIS. We used (99m)Tc pertechnetate as a radiotracer for SPECT/CT imaging to determine the pattern of ectopic NIS expression in longitudinal kinetic studies. Some animals of the cohort were culled and NIS expression was measured by quantitative RT-PCR and immunohistochemistry. The radioactive content of some liver biopsies was also measured ex vivo. Our results show that in longitudinal studies involving datasets taken from individual mice, the presentation of non-normalised data (activity expressed as %ID/g or %ID/cc) leads to 'noisy', and sometimes incoherent, results. This variability is due to the fact that the blood pertechnetate concentration can vary up to three-fold from day to day. Normalisation of these data with blood activities corrects for these inconsistencies. We advocate that, blood pertechnetate activity should be determined and used to normalise the activity measured in the organ/region of interest that expresses NIS ectopically. Considering that NIS imaging has already reached the clinical setting in the context of cancer gene therapy, this normalisation may be essential in order to obtain accurate and predictive information in future longitudinal clinical studies in biotherapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0034086PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3309932PMC
August 2012

Revisiting iodination sites in thyroglobulin with an organ-oriented shotgun strategy.

J Biol Chem 2011 Jan 26;286(1):259-69. Epub 2010 Oct 26.

Commissariat à l'Energie Atomique, DSV, iBEB, Laboratoire des Transporters en Imagerie et Radiothérapie en Oncologie, Bagnols-sur-Cèze F-30207, France.

Thyroglobulin (Tg) is secreted by thyroid epithelial cells. It is essential for thyroid hormonogenesis and iodine storage. Although studied for many years, only indirect and partial surveys of its post-translational modifications were reported. Here, we present a direct proteomic approach, used to study the degree of iodination of mouse Tg without any preliminary purification. A comprehensive coverage of Tg was obtained using a combination of different proteases, MS/MS fragmentation procedures with inclusion lists and a hybrid mass high-resolution LTQ-Orbitrap XL mass spectrometer. Although only 16 iodinated sites are currently known for human Tg, we uncovered 37 iodinated tyrosine residues, most of them being mono- or diiodinated. We report the specific isotopic pattern of thyroxine modification, not recognized as a normal peptide pattern. Four hormonogenic sites were detected. Two donor sites were identified through the detection of a pyruvic acid residue in place of the initial tyrosine. Evidence for polypeptide cleavages sites due to the action of cathepsins and dipeptidyl proteases in the thyroid were also detected. This work shows that semi-quantitation of Tg iodination states is feasible for human biopsies and should be of significant medical interest for further characterization of human thyroid pathologies.
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http://dx.doi.org/10.1074/jbc.M110.159483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012983PMC
January 2011

Characterisation of the purified human sodium/iodide symporter reveals that the protein is mainly present in a dimeric form and permits the detailed study of a native C-terminal fragment.

Biochim Biophys Acta 2011 Jan 23;1808(1):65-77. Epub 2010 Aug 23.

CEA, iBEB, SBTN, Centre de Marcoule, Bat 170, BP17171, 30207 Bagnols sur Cèze, CEDEX, France.

The sodium/iodide symporter is an intrinsic membrane protein that actively transports iodide into thyroid follicular cells. It is a key element in thyroid hormone biosynthesis and in the radiotherapy of thyroid tumours and their metastases. Sodium/iodide symporter is a very hydrophobic protein that belongs to the family of sodium/solute symporters. As for many other membrane proteins, particularly mammalian ones, little is known about its biochemistry and structure. It is predicted to contain 13 transmembrane helices, with an N-terminus oriented extracellularly. The C-terminal, cytosolic domain contains approximately one hundred amino acid residues and bears most of the transporter's putative regulatory sites (phosphorylation, sumoylation, di-acide, di-leucine or PDZ-binding motifs). In this study, we report the establishment of eukaryotic cell lines stably expressing various human sodium/iodide symporter recombinant proteins, and the development of a purification protocol which allowed us to purify milligram quantities of the human transporter. The quaternary structure of membrane transporters is considered to be essential for their function and regulation. Here, the oligomeric state of human sodium/iodide symporter was analysed for the first time using purified protein, by size exclusion chromatography and light scattering spectroscopy, revealing that the protein exists mainly as a dimer which is stabilised by a disulfide bridge. In addition, the existence of a sodium/iodide symporter C-terminal fragment interacting with the protein was also highlighted. We have shown that this fragment exists in various species and cell types, and demonstrated that it contains the amino-acids [512-643] from the human sodium/iodide symporter protein and, therefore, the last predicted transmembrane helix. Expression of either the [1-512] truncated domain or the [512-643] domain alone, as well as co-expression of the two fragments, was performed, and revealed that co-expression of [1-512] with [512-643] allowed the reconstitution of a functional protein. These findings constitute an important step towards an understanding of some of the post-translational mechanisms that finely tune iodide accumulation through human sodium/iodide symporter regulation.
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http://dx.doi.org/10.1016/j.bbamem.2010.08.013DOI Listing
January 2011

Distribution and dynamics of (99m)Tc-pertechnetate uptake in the thyroid and other organs assessed by single-photon emission computed tomography in living mice.

Thyroid 2010 May;20(5):519-26

Transporters, Imaging and Radiotheraphy in Oncology (TIRO), Commissariat Energie Atomique Direction des Sciences du Vivant (DSV), Institut de Biologie Environementale et Biotechnologie (iBEB), Service de Biochimie et Toxicologie Nucléaire (SBTN), Centre Antoine Lacassagne, University of Nice Sophia Antipolis , Nice, France .

Background: (99m)Tc pertechnetate is a well-known anion, used for clinical imaging of thyroid function. This gamma emitter is transported by the sodium iodide symporter but is not incorporated into thyroglobulin. Scintigraphy using (99m)Tc pertechnetate or (123)iodide represents a powerful tool for the study of sodium iodide symporter activity in different organs of living animal models. However, in many studies that have been performed in mice, the thyroid could not be distinguished from the salivary glands. In this work, we have evaluated the use of a clinically dedicated single-photon emission computed tomography (SPECT) camera for thyroid imaging and assessed what improvements are necessary for the development of this technique.

Methods: SPECT of the mouse neck region, with pinhole collimation and geometric calibration, was used for the individual measurement of (99m)Tc pertechnetate uptake in the thyroid and the salivary glands. Uptake in the stomach was studied by planar whole-body imaging. Uptake kinetics and biodistribution studies were performed by sequential imaging.

Results: This work has shown that thyroid imaging in living mice can be performed with a SPECT camera originally built for clinical use. Our experiments indicate that (99m)Tc pertechnetate uptake is faster in the thyroid than in the salivary glands and the stomach. The decrease in (99m)Tc pertechnetate uptake after injection of iodide or perchlorate as competitive inhibitors was also studied. The resulting rate decreases were faster in the thyroid than in the salivary glands or the stomach.

Conclusions: We have shown that a clinically dedicated SPECT camera can be used for thyroid imaging. In our experiments, SPECT imaging allowed the analysis of (99m)Tc pertechnetate accumulation in individual organs and revealed differences in uptake kinetics.
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http://dx.doi.org/10.1089/thy.2009.0213DOI Listing
May 2010

Characterization of small-molecule inhibitors of the sodium iodide symporter.

J Endocrinol 2009 Mar 9;200(3):357-65. Epub 2008 Dec 9.

TIRO, CEA DSV-iBEB-SBTN, CAL, School of Medicine, University of Nice Sophia Antipolis, Nice, France.

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.
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http://dx.doi.org/10.1677/JOE-08-0246DOI Listing
March 2009

Immunoanalysis indicates that the sodium iodide symporter is not overexpressed in intracellular compartments in thyroid and breast cancers.

Eur J Endocrinol 2009 Feb 24;160(2):215-25. Epub 2008 Nov 24.

Department of Pathology, Center Antoine Lacassagne, 06100, Nice, France.

Objective: The active transport of iodide into thyroid cells is mediated by the Na(+)/I(-) symporter (NIS) located in the basolateral membrane. Strong intracellular staining with anti-NIS antibodies has been reported in thyroid and breast cancers. Our initial objective was to screen tumour samples for intracellular NIS staining and then to study the mechanisms underlying the altered subcellular localization of the transporters.

Methods: Immunostaining using three different anti-NIS antibodies was performed on paraffin-embedded tissue sections from 93 thyroid or breast cancers. Western blot experiments were carried out to determine the amount of NIS protein in 20 samples.

Results: Using three different anti-NIS antibodies, we observed intracellular staining in a majority of thyroid tumour samples. Control immunohistochemistry and western blot experiments indicated that this intracellular staining was due to non-specific binding of the antibodies. In breast tumours, very weak intracellular staining was observed in some samples. Western blot experiments suggest that this labelling is also non-specific.

Conclusions: Our results strongly indicate that the NIS protein level is low in thyroid and breast cancers and that the intracellular staining obtained with anti-NIS antibodies corresponds to a non-specific signal. Accordingly, to increase the efficiency of radiotherapy for thyroid cancers and to enable the use of radioiodine in the diagnosis and therapy of breast tumours, improving NIS targeting to the plasma membrane will not be sufficient. Instead, increasing the expression level of NIS should remain the major goal of this field.
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http://dx.doi.org/10.1530/EJE-08-0505DOI Listing
February 2009

Comparison of expressed human and mouse sodium/iodide symporters reveals differences in transport properties and subcellular localization.

J Endocrinol 2008 Apr;197(1):95-109

TIRO, CEA DSV-iBEB-SBTN, CAL, School of Medicine, University of Nice Sophia Antipolis, 28 Avenue de Valombrose, 06107, Nice, France.

The active transport of iodide from the bloodstream into thyroid follicular cells is mediated by the Na+/I- symporter (NIS). We studied mouse NIS (mNIS) and found that it catalyzes iodide transport into transfected cells more efficiently than human NIS (hNIS). To further characterize this difference, we compared (125)I uptake in the transiently transfected human embryonic kidney (HEK) 293 cells. We found that the V(max) for mNIS was four times higher than that for hNIS, and that the iodide transport constant (K(m)) was 2.5-fold lower for hNIS than mNIS. We also performed immunocytolocalization studies and observed that the subcellular distribution of the two orthologs differed. While the mouse protein was predominantly found at the plasma membrane, its human ortholog was intracellular in approximately 40% of the expressing cells. Using cell surface protein-labeling assays, we found that the plasma membrane localization frequency of the mouse protein was only 2.5-fold higher than that of the human protein, and therefore cannot alone account for the difference in the obtained V(max) values. We reasoned that the observed difference could also be caused by a higher turnover number for iodide transport in the mouse protein. We then expressed and analyzed chimeric proteins. The data obtained with these constructs suggest that the iodide recognition site could be located in the region extending from the N-terminus to transmembrane domain 8, and that the region between transmembrane domain 5 and the C-terminus could play a role in the subcellular localization of the protein.
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http://dx.doi.org/10.1677/JOE-07-0455DOI Listing
April 2008

Small-molecule inhibitors of sodium iodide symporter function.

Chembiochem 2008 Apr;9(6):889-95

Department of Bioorganic Chemistry and Isotopic Labelling, CEA, Institute of Biology and Technology (iBiTecS), Gif-sur-Yvette 91191, France.

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.
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http://dx.doi.org/10.1002/cbic.200700682DOI Listing
April 2008

A 96-well automated radioiodide uptake assay for sodium/iodide symporter inhibitors.

Assay Drug Dev Technol 2007 Aug;5(4):535-40

iBiTecS, Service de Chimie Bioorganique et de Marquage, CEA, Gif sur Yvette, France.

A high-throughput screening method based on radioiodide uptake in human embryonic kidney 293 cells expressing the human sodium/iodide symporter was developed. Central to assay development was a homogeneous cell culture in the 96-well microplate coupled with the use of scintillation proximity technology. The assay is fast and highly reproducible with a Z' greater than 0.8. The automated procedure allows the screening of 4,000 compounds per day. Using this methodology, several known substrates of the sodium/iodide symporter were evaluated in a single day. Inhibition of iodide uptake was shown to follow the series PF(6)(-) > ClO(4)(-) > BF(4)(-) > SCN(-) > NO(3)(-) > IO(4)(-) > N(3)(-) > Br(-), in accord with the literature. This method represents an initial approach to the search for inhibitors of iodide transport mediated by the sodium/iodide symporter.
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http://dx.doi.org/10.1089/adt.2007.068DOI Listing
August 2007

Sugar binding induced charge translocation in the melibiose permease from Escherichia coli.

Biochemistry 2004 Oct;43(39):12606-13

Max-Planck-Institut für Biophysik, Marie-Curie Strasse 15, 60439 Frankfurt/M, Germany.

Electrogenic events associated with the activity of the melibiose permease (MelB), a transporter from Escherichia coli, were investigated. Proteoliposomes containing purified MelB were adsorbed to a solid supported lipid membrane, activated by a substrate concentration jump, and transient currents were measured. When the transporter was preincubated with Na(+) at saturating concentrations, a charge translocation in the protein upon melibiose binding could still be observed. This result demonstrates that binding of the uncharged substrate melibiose triggers a charge displacement in the protein. Further analysis showed that the charge displacement is neither related to extra Na(+) binding to the transporter, nor to the displacement of already bound Na(+) within the transporter. The electrogenic melibiose binding process is explained by a conformational change with concomitant displacement of charged amino acid side chains and/or a reorientation of helix dipoles. A kinetic model is suggested, in which Na(+) and melibiose binding are distinct electrogenic processes associated with approximately the same charge displacement. These binding reactions are fast in the presence of the respective cosubstrate (k > 50 s(-1)).
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http://dx.doi.org/10.1021/bi0489053DOI Listing
October 2004