NIS Gene Expression Research Articles

Ammonium perchlorate: serum dosimetry, neurotoxicity, and resilience of the neonatal rat thyroid system.

The environmental contaminant perchlorate impairs the synthesis of thyroid hormones by reducing iodine uptake into the thyroid gland. Despite this known action, moderate doses of perchlorate do not significantly alter serum thyroid hormone in rat pups born to exposed dams. We examined perchlorate dosimetry and responsivity of the thyroid gland and brain in offspring following maternal exposure to perchlorate. Pregnant rat dams were delivered perchlorate in drinking water (0, 30, 100, 300, 1000 ppm) from gestational day 6 to postnatal day (PN) 21. Perchlorate was present in the placenta, milk, and serum, the latter declining in pups over the course of lactation. Serum and brain thyroid hormone were reduced in pups at birth but recovered to control levels by PN2. Dramatic upregulation of Nis was observed in the thyroid gland of the exposed pup. Despite the return of serum thyroid hormone to control levels by PN2, expression of several TH-responsive genes was altered in the PN14 pup brain. Contextual fear learning was unimpaired in the adults, supporting previous reports. Declining levels of serum perchlorate and a profound upregulation of Nis gene expression in the thyroid gland are consistent with the rapid return to the euthyroid state in the neonate. However, despite this recovery, thyroid hormone insufficiencies in serum and brain beginning in utero and present at birth appear sufficient to alter TH action in the fetus and subsequent trajectory of brain development. Biomarkers of that altered trajectory remain in the brain of the neonate, demonstrating that perchlorate is not devoid of effects on the developing brain.

Analysis of NIS Plasma Membrane Interactors Discloses Key Regulation by a SRC/RAC1/PAK1/PIP5K/EZRIN Pathway with Potential Implications for Radioiodine Re-Sensitization Therapy in Thyroid Cancer.

Simple SummaryThe presence of the sodium–iodide symporter (NIS) at the plasma membrane (PM) of differentiated thyroid cancer (DTC) cells is required for the successful use of radioiodine (RAI) therapy in these malignancies. However, NIS is frequently downregulated in malignant thyroid tissue, and 30% to 50% of metastatic DTCs become refractory to RAI treatment. Notably, many RAI-refractory DTCs still show relatively abundant NIS protein levels despite failing to uptake sufficient iodide to enable RAI therapy. This suggests an impairment in the NIS delivery and retention at the PM. Because little is known about these mechanisms, we employed a new experimental strategy to purify and characterize proteins which are selectively associated with NIS at the PM. We found that, when at the PM, NIS is particularly associated with proteins that regulate the cell’s actin cytoskeleton. Further investigation revealed that several of these proteins integrate an intracellular signaling pathway that modulates the functional localization of NIS at the PM, in normal and DTC cells. Our findings open new venues for therapeutic intervention in refractory DTC, envisioning the re-sensitization of these tumors to RAI therapy.The functional expression of the sodium–iodide symporter (NIS) at the membrane of differentiated thyroid cancer (DTC) cells is the cornerstone for the use of radioiodine (RAI) therapy in these malignancies. However, NIS gene expression is frequently downregulated in malignant thyroid tissue, and 30% to 50% of metastatic DTCs become refractory to RAI treatment, which dramatically decreases patient survival. Several strategies have been attempted to increase the NIS mRNA levels in refractory DTC cells, so as to re-sensitize refractory tumors to RAI. However, there are many RAI-refractory DTCs in which the NIS mRNA and protein levels are relatively abundant but only reduced levels of iodide uptake are detected, suggesting a posttranslational failure in the delivery of NIS to the plasma membrane (PM), or an impaired residency at the PM. Because little is known about the molecules and pathways regulating NIS delivery to, and residency at, the PM of thyroid cells, we here employed an intact-cell labeling/immunoprecipitation methodology to selectively purify NIS-containing macromolecular complexes from the PM. Using mass spectrometry, we characterized and compared the composition of NIS PM complexes to that of NIS complexes isolated from whole cell (WC) lysates. Applying gene ontology analysis to the obtained MS data, we found that while both the PM-NIS and WC-NIS datasets had in common a considerable number of proteins involved in vesicle transport and protein trafficking, the NIS PM complexes were particularly enriched in proteins associated with the regulation of the actin cytoskeleton. Through a systematic validation of the detected interactions by co-immunoprecipitation and Western blot, followed by the biochemical and functional characterization of the contribution of each interactor to NIS PM residency and iodide uptake, we were able to identify a pathway by which the PM localization and function of NIS depends on its binding to SRC kinase, which leads to the recruitment and activation of the small GTPase RAC1. RAC1 signals through PAK1 and PIP5K to promote ARP2/3-mediated actin polymerization, and the recruitment and binding of the actin anchoring protein EZRIN to NIS, promoting its residency and function at the PM of normal and TC cells. Besides providing novel insights into the regulation of NIS localization and function at the PM of TC cells, our results open new venues for therapeutic intervention in TC, namely the possibility of modulating abnormal SRC signaling in refractory TC from a proliferative/invasive effect to the re-sensitization of these tumors to RAI therapy by inducing NIS retention at the PM.

FOXA1 Regulation Turns Benzamide HDACi Treatment Effect-Specific in BC, Promoting NIS Gene-Mediated Targeted Radioiodine Therapy

Human sodium iodide symporter (NIS) gene mediated radio-ablation is a successful procedure in thyroid cancer clinics. In recent years, natural expression of NIS is reported in breast cancer (BC) cases but is yet to make its mark as a therapeutic procedure in BC clinics. A pre-exposure to histone deacetylase (HDAC) inhibitors to amplify endogenous NIS expression was attempted, but achieving cancer tissue-specific enhancement of NIS in patients is an important challenge to win. Here, for the first time, we show that a benzamide class of HDACi (bHDACi) can significantly induce NIS gene expression and function (p < 0.05) in BC cells with minimal off-target effects. Transcription factor (TF) profiler and promoter binding array reveals 22 TFs differentially activated by CI-994, of which FOXA1 is identified as a unique and positive regulator of NIS. Clonogenic assay shows reduced survival with bHDACi + 131I combination treatment. Further, AR-42 and MS-275 treatment shows enhanced NIS expression in an orthotopic breast tumor model. Combining bHDACi with 1 mCi 131I shows 40% drop in signal (p < 0.05), indicating enhanced radio-ablation effect. Cerenkov imaging revealed higher accumulation of 131I in MS-275-treated tumors. Thus, bHDACi-mediated selective enhancement ensuring minimal off-target effect is a step further toward using NIS as a therapeutic target for BC.

Thyrotropin, but Not Thyroid-Stimulating Antibodies, Induces Biphasic Regulation of Gene Expression in Human Thyrocytes.

Background: Thyrotropin (TSH) and thyroid-stimulating antibodies (TSAbs) activate TSH receptor (TSHR) signaling by binding to its extracellular domain. TSHR signaling has been studied extensively in animal thyrocytes and in engineered cell lines, and differences in signaling have been observed in different cell systems. We, therefore, decided to characterize and compare TSHR signaling mediated by TSH and monoclonal TSAbs in human thyrocytes in primary culture. Methods: We used quantitative reverse transcription-polymerase chain reaction to measure mRNA levels of thyroid-specific genes thyroglobulin (TG), thyroperoxidase (TPO), iodothyronine deiodinase type 2 (DIO2), sodium-iodide symporter (NIS), and TSHR after stimulation by TSH or two monoclonal TSAbs, KSAb1 and M22. We also compared secreted TG protein after TSHR activation by TSH and TSAbs using an enzyme-linked immunosorbent assay. TSHR cell surface expression was determined using fluorescence activated cell sorting (FACS). Results: We found that TSH at low doses increases and at high doses (>1 mU/mL) decreases levels of gene expression for TSHR, TG, TPO, NIS, and DIO2. The biphasic effect of TSH on signaling was not caused by downregulation of cell surface TSHRs. This bell-shaped biphasic dose-response curve has been termed an inverted U-shaped dose-response curve (IUDRC). An IUDRC was also found for TSH-induced regulation of TG secretion. In contrast, KSAb1- and M22-induced regulation of TSHR, TG, TPO, NIS, and DIO2 gene expression, and secreted TG followed a monotonic dose-response curve that plateaus at high doses of activating antibody. Conclusions: Our data demonstrate that the physiological activation of TSHRs by TSH in primary cultures of human thyrocytes is characterized by a regulatory mechanism that may inhibit thyrocyte overstimulation. In contrast, TSAbs do not exhibit biphasic regulation. Although KSAb1 and M22 may not be representative of all TSAbs found in patients with Graves' disease, we suggest that persistent robust stimulation of TSHRs by TSAbs, unrelieved by a decrease at high TSAb levels, fosters chronic stimulation of thyrocytes in Graves' hyperthyroidism.

Targeting the BRAF Signaling Pathway in CD133pos Cancer Stem Cells of Anaplastic Thyroid Carcinoma

Background:Cancer stem cells (CSCs) with a self-renewal ability in tumor cells population, execute a pivotal function in tumorigenesis, retrogression, and metastasis of malignant cancers such as anaplastic thyroid carcinoma (ATC).Materials and Methods:In this study, we isolated CSCs subpopulation with CD133 surface marker from three ATC cell lines by magnetic cell sorting assay. After confirming the segregation by the flow cytometry method, BRAF and sodium-iodide symporter (NIS) genes were investigated in them before and after incubation with BRAF inhibitor. Also, we evaluated the NIS protein expression and localization.Results:Established upon q-RT PCR data, when compared to human normal thyrocytes, the BRAFV600E gene was over-expressed in CD133pos cells (>1705.99 ± 55.55 fold, Mean ± SEM, n=3, P- value<0.05), whilst the expression of NIS gene was very restricted (< 0.0008 ± 5.43 fold, Mean ± SEM, n=3, P- value<0.05) in them. Also, our results showed that BRAF inhibition affected NIS protein expression and localization.Conclusions:Current study showed that the differentiate genes/proteins expression can be induced in the CSCs via focus on signal transduction pathways and targeting their molecules, that are involved in expression of these genes/proteins. Therefore, attention to targeting CSCs along with routine thyroid cancer therapy, can help to ATC treatment.

Dual-targeted NIS polyplexes-a theranostic strategy toward tumors with heterogeneous receptor expression.

Tumor heterogeneity, within and between tumors, may have severe implications for tumor therapy, especially for targeted gene therapy, where single-targeted approaches often result in limited efficacy and therapy resistance. Polymer-formulated nonviral vectors provide a potent delivery platform for cancer therapy. To improve applicability for future clinical use in a broad range of patients and cancer types, a dual-targeting approach was performed. Synthetic LPEI-PEG2kDa-based polymer backbones were coupled to two tumor-specific peptide ligands GE11 (EGFR-targeting) and cMBP (cMET-targeting). The dual-targeting approach was used to deliver the theranostic sodium iodide symporter (NIS) gene to hepatocellular cancer. NIS as auspicious theranostic gene allows noninvasive imaging of functional NIS gene expression and effective anticancer radioiodide therapy. Enhanced tumor-specific transduction efficiency of dual-targeted polyplexes compared to single-targeted polyplexes was demonstrated in vitro using tumor cell lines with different EGFR and cMET expression and in vivo by 124I-PET-imaging. Therapeutic efficacy of the bispecific concept was mirrored by significantly reduced tumor growth and perfusion, which was associated with prolonged animal survival. In conclusion, the dual-targeting approach highlights the benefits of a bifunctional strategy for a future clinical translation of the bioimaging-based NIS-mediated radiotherapy allowing efficient targeting of heterogeneic tumors with variable receptor expression levels.

In Vivo Estimation of Oncolytic Virus Populations within Tumors.

The use of replication-competent viruses as oncolytic agents is rapidly expanding, with several oncolytic viruses approved for cancer therapy. As responses to therapy are highly variable, understanding the dynamics of therapy is critical for optimal application of virotherapy in practice. Although mathematical models have been developed to understand the dynamics of tumor virotherapy, a scarcity of in vivo data has made difficult parametrization of these models. To tackle this problem, we studied the in vitro and in vivo spread of two oncolytic measles viruses that induce expression of the sodium iodide symporter (NIS) in cells. NIS expression enabled infected cells to concentrate radioactive isotopes that could be reproducibly and quantitatively imaged using SPECT/CT. We observed a strong linear relationship in vitro between infectious virus particles, viral N and NIS gene expression, and radioactive isotope uptake. In vivo radioisotope uptake was highly correlated with viral N and NIS gene expression. Similar expression patterns between viral N and NIS gene expression in vitro and in vivo implied that the oncolytic virus behaved similarly in both scenarios. Significant titers of viable virus were consistently isolated from tumors explanted from mice that had been injected with oncolytic measle viruses. We observed a weaker but positive in vivo relationship between radioisotope uptake and the viable virus titer recovered from tumors; this was likely due to anisotropies in the viral distribution in vivo These data suggest that methods that enable quantitation of in vivo anisotropies are required for continuing development of oncolytic virotherapy.Significance: These findings address a fundamental gap in our knowledge of oncolytic virotherapy by presenting technology that gives insight into the behavior of oncolytic viruses in vivo Cancer Res; 78(20); 5992-6000. ©2018 AACR.

An investigation of NIS expression in gastric tissue of obese individuals

Obesity is seen as one of the top ten (10) illness's listed by World Health Organization (WHO). It is a global problem that can affect people of all ages. Obesity is identified one of the most important factors leading to diabetes, heart disease and hypertension. Individuals with a Body Mass Index (BMI) above 40 kg/m2 are defined with morbid obesity. Sodium Iodide Symporter (NIS) gene is a plasma membrane glycoprotein that mediates iodide uptake in thyroid glands, stomach, salivary glands, lactating mammary glands and intestine. NIS gene transports iodide from the blood to the gastric epithelial cells. NIS gene expression and regulatory role of NIS gene in gastrointestinal tract, hasn't been studied yet in the individuals with obesity (i.e., BMI &gt;40 kg/m2). In this study, gastric tissues were obtained by laparoscopic sleeve gastrectomy from 33 individuals diagnosed with obesity. Control group consisted of gastric tissue of 21 subjects with normal BMI obtained by endoscopy. RNA isolation, cDNA synthesis and qRT-PCR analyses were performed on the samples to determine NIS gene expression. Expression levels of NIS gene were compared between obese and control individuals, although an increase was observed in obese patients this difference was not found to be statistically important (p&gt;0.05).

Molecular Profiling of Follicular Variant of Papillary Thyroid Cancer Reveals Low-Risk Noninvasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features: A Paradigm Shift to Reduce Aggressive Treatment of Indolent Tumors.

Introduction:Encapsulated follicular variant of papillary thyroid carcinoma (EFVPTC) has been reclassified into noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) and invasive EFVPTC. NIFTP is considered a low-risk neoplasm. Therefore, follicular variant of papillary thyroid cancer (FVPTC) presently has two distinct histopathological subtypes – invasive EFVPTC and infiltrative/diffuse FVPTC. Molecular characteristics of these groups remain unclear.Methodology:Thirty FVPTCs (10 NIFTPs, 12 invasive EFVPTCs, and 8 infiltrative/diffuse variants) were reviewed and screened for BRAF and RAS mutations by restriction fragment length morphism-polymerase chain reaction (PCR) and Sanger sequencing. The mRNA expression levels of iodine-metabolizing genes were analyzed using real-time PCR. The mutations status and mRNA expression levels were correlated with clinicopathological features.Results:All 10 NIFTPs had predominant follicular pattern. One case showed NRAS mutation, whereas none showed BRAF mutation. All invasive EFVPTC had capsular and/or lymphovascular invasion and 4/12 showed lymph node metastasis. BRAF and NRAS were seen in three cases each of invasive FVPTC. All eight infiltrating/diffuse FVPTCs showed infiltration into adjacent thyroid parenchyma and lymph node metastasis.Conclusion:BRAF mutation was observed in 62.5% of cases; however, no NRAS mutation was found. Sodium iodide symporter (NIS) expressions in NIFTP were similar to that of normal thyroid tissue, whereas it was downregulated in invasive and infiltrative/diffuse FVPTC. Our study supports the argument that NIFTP can be considered as low-risk follicular thyroid neoplasm. Those tumors that harbor BRAF mutations may be offered a complete thyroidectomy because they show decreased expression of NIS gene which confers a tendency to lose radioactive iodine avidity and further recurrence of the tumor.

EGFR Targeting and Shielding of pDNA Lipopolyplexes via Bivalent Attachment of a Sequence-Defined PEG Agent.

For successful nonviral gene delivery, cationic polymers are promising DNA carrier, which need to comprise several functionalities. The current work focuses on the postincorporation of epidermal growth factor receptor (EGFR) targeted PEGylation agents onto lipopolyplexes for pDNA delivery. T-shaped lipo-oligomers are previously found to be effective sequence-defined carriers for pDNA and siRNA. Here, the bis-oleoyl-oligoaminoethanamide 454 containing tyrosine trimer-cysteine ends is applied for complex formation with pDNA coding for luciferase or sodium iodide symporter (NIS). In a second step, the lipopolyplexes are modified via disulfide formation with sequence-defined monovalent or bivalent PEGylation agents containing one or two 3-nitro-2-pyridinesulfenyl (NPys)-activated cysteines, respectively. For targeting, the polyethylene glycol (PEG) agents comprise the EGFR targeting peptide GE11. In comparison of all transfection complexes, 454 lipopolyplexes modified with the bidentate PEG-GE11 agent show the best, EGFR-dependent uptake as well as luciferase and NIS gene expression into receptor-positive tumor cells.

Supplying rapeseed meal to the diets with or without potassium iodide for yellow catfish (Tachysurus fulvidraco)

The aims of the study were (1) to determine the effect of rapeseed meal on growth, body composition, thyroid hormones, and expressions of three genes involved in thyroid function and (2) to determine if potassium iodide in the diet counteracts the adverse effect caused by rapeseed meal on growth. One fish meal-based diet was serviced as the control. The other six diets were designed on the basis of 3 × 2 factorial design. The rapeseed meal inclusion (10, 20, and 30%) and potassium iodide inclusion (0 and 72 mg (kg)−1) were set to be two factors. The fish experiment was designed according to a randomized block model with three size groups of fish (27.7, 19.9, and 8.1 g). The feeding period lasted for 71 days. The results showed that final weight, weight gain, specific growth rate (SGR), and feed conversion ratio (FCR) of fish fed with 10% rapeseed meal-based diets were significantly better than those fed with 20 and 30% rapeseed meal. The highest nitrogen retention and protein efficiency ratio (PER) were in the 10% rapeseed group. Inclusion of 30% rapeseed meal in the diets significantly reduced whole-fish dry matter, Ile, and Phe contents. The gene expressions of the thyrotropin receptor (TSHR), sodium/iodide symporter (NIS), and thyroid peroxidase (TPO), as well as the thyroid hormone (T3 and T4) levels in the plasma, were not affected by the different diets. However, TSHR and TPO gene expressions negatively correlated with T4 and lipid retention. NIS gene expression positively correlated with cumulative feed intake, final weight, weight gain, SGR, nitrogen retention, and PER. It also negatively correlated with FCR. In conclusion, the rapeseed meal inclusion level in the diet was suggested to be 10%. Potassium iodide in the diet (72 mg (kg)−1) did not counteract the adverse effects caused by rapeseed meal on fish growth. Correlations provided evidence to support the link between NIS gene and fish growth.

A Novel Ideal Radionuclide Imaging System for Non-invasively Cell Monitoring built on Baculovirus Backbone by Introducing Sleeping Beauty Transposon

Sleeping Beauty (SB) transposon is an attractive tool in stable transgene integration both in vitro and in vivo; and we introduced SB transposon into recombinant sodium-iodide symporter baculovirus system (Bac-NIS system) to facilitate long-term expression of recombinant sodium-iodide symporter. In our study, two hybrid baculovirus systems (Bac-eGFP-SB-NeoR and Bac-NIS-SB-NeoR) were successfully constructed and used to infect U87 glioma cells. After G418 selection screening, the Bac-eGFP-SB-NeoR-U87 cells remained eGFP positive, at the 18th and 196th day post transfection (96.03 ± 0.21% and 97.43 ± 0.81%), while eGFP positive population declined significantly at 18 days in cells transfected with unmodified baculovirus construct. NIS gene expression by Bac-NIS-SB-NeoR-U87 cells was also maintained for 28 weeks as determined by radioiodine uptake assay, reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot (WB) assay. When transplanted in mice, Bac-NIS-SB-NeoR-U87 cells also expressed NIS gene stably as monitored by SPECT imaging for 43 days until the tumor-bearing mice were sacrificed. Herein, we showed that incorporation of SB in Bac-NIS system (hybrid Bac-NIS-SB-NeoR) can achieve a long-term transgene expression and can improve radionuclide imaging in cell tracking and monitoring in vivo.

Effect of excess iodine intake on thyroid on human health.

The recommended daily intake of iodide, is 150 μg for adolescents and adults, 250 μg for pregnancy and lactation. Thyroid gland is an effective collector of iodine. The active iodine uptake along the basolateral membrane of thyroid cell is followed by its transport to the apical edge of the cell and then to the follicle lumen. TSH acts through cAMP and stimulates NIS gene expression and protein synthesis. The major proportion of iodine in the thyroid gland is bound to Thyroglobulin. The non-organic intrathyroidal iodine is usually low, but significantly greater compared to plasma. Large doses of iodine reduce both the uptake and the organification (Wolff-Chaikoff effect) and cause partial inhibition of Tg proteolysis. The thyroid gland has several protective mechanisms resulting on the maintenance of normal thyroid function despite wide fluctuations of the daily iodine intake. Ingestion of several commonly used drugs and food conservatives results in acute or chronic excessive iodine intake. Failure to escape from the iodine induced organification inhibition can cause hypothyroidism, which is temporary and subsides after iodine exposure ceases. Iodine excess may also establish a status of excessive thyroid hormone synthesis and release, thus inducing autonomic thyroid function in iodopenic areas or can contribute to the development of iodine-induced hyperthyroidism in iodine abundant areas. The anti-arrhythmic Amiodarone, is a benzofuranic product with a very high iodine content, is associated with either hypo- or hyperthyroidism development. In the presence of defective auto-protective mechanisms, excessive iodine ingestion can divert the normal thyroid function.

Clinical Manifestations and Gene Expression in Patients with Conventional Papillary Thyroid Carcinoma Carrying the BRAF(V600E) Mutation and BRAF Pseudogene.

The association of BRAF(V600E) with the clinical manifestations of papillary thyroid carcinoma (PTC) remains controversial. Recent studies have shown that the BRAF pseudogene can activate the MAPK pathway and induce tumorigenesis. This study investigated the association of BRAF(V600E), the BRAF pseudogene, and their mRNA levels with clinical features and thyroid-specific gene expression in conventional PTCs. A total of 78 specimens were collected from patients with conventional PTCs. RNA was isolated, and quantitative polymerase chain reaction was used to measure the mRNA levels of BRAF, the BRAF pseudogene, and thyroid-specific and tumor-related genes. Immunohistochemical (IHC) staining of BRAF, ERK, sodium-iodide symporter (NIS), thyrotropin receptor, glucose transporter 1, and Ki67 was also performed. BRAF(V600E) and the BRAF pseudogene were detected in 73.0% (57/78) and 91.7% (44/48), respectively, of the conventional PTCs. The presence of BRAF(V600E) was not associated with the multiple clinical features assessed or the recurrence rate during 76.9 ± 47.2 months of follow-up. Neither was it associated with IHC staining or tumor-related/thyroid-specific gene expression, except for decreased NIS gene expression. The BRAF pseudogene was not associated with clinical characteristics or thyroid-specific gene expression, except for decreased decoy receptor 3 (DCR3) expression. High BRAF mRNA levels were associated with bilateral and multifocal lesions, and BRAF-pseudogene mRNA levels were positively correlated with BRAF mRNA levels (r = 0.415, p = 0.009). These results do not support the use of the BRAF(V600E) mutation as a prognostic marker of conventional PTC. However, the association of high BRAF mRNA levels with more advanced clinical features suggests that BRAF mRNA levels might be a more useful clinical marker of PTCs, independent of the BRAF(V600E) mutation status. The correlation between BRAF-pseudogene mRNA levels and BRAF mRNA levels in PTCs is in agreement with the hypothesis that the BRAF pseudogene regulates BRAF expression during tumorigenesis by acting as competitive noncoding RNA. However, additional studies with larger sample sizes are required to confirm these findings.

Excess iodide downregulates Na+/I− symporter gene transcription through activation of PI3K/Akt pathway

Transcriptional mechanisms associated with iodide-induced downregulation of NIS expression remain uncertain. Here, we further analyzed the transcriptional regulation of NIS gene expression by excess iodide using PCCl3 cells. NIS promoter activity was reduced in cells treated for 12–24 h with 10−5 to 10−3 M NaI. Site-directed mutagenesis of Pax8 and NF-κB cis-acting elements abrogated the iodide-induced NIS transcription repression. Indeed, excess iodide (10−3 M) excluded Pax8 from the nucleus, decreased p65 total expression and reduced their transcriptional activity. Importantly, p65-Pax8 physical interaction and binding to NIS upstream enhancer were reduced upon iodide treatment. PI3K/Akt pathway activation by iodide-induced ROS production is involved in the transcriptional repression of NIS expression. In conclusion, the results indicated that excess iodide transcriptionally represses NIS gene expression through the impairment of Pax8 and p65 transcriptional activity. Furthermore, the data presented herein described novel roles for PI3K/Akt signaling pathway and oxidative status in the thyroid autoregulatory phenomenon.

Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer

Oncolytic strains of vaccinia virus are currently in clinical development with clear evidence of safety and promising signs of efficacy. Addition of therapeutic genes to the viral genome may increase the therapeutic efficacy of vaccinia. We evaluated the therapeutic potential of vaccinia virus expressing the sodium iodide symporter (NIS) in prostate cancer models, combining oncolysis, external beam radiotherapy and NIS-mediated radioiodide therapy. The NIS-expressing vaccinia virus (VV-NIS), GLV-1h153, was tested in in vitro analyzes of viral cell killing, combination with radiotherapy, NIS expression, cellular radioiodide uptake and apoptotic cell death in PC3, DU145, LNCaP and WPMY-1 human prostate cell lines. In vivo experiments were carried out in PC3 xenografts in CD1 nude mice to assess NIS expression and tumor radioiodide uptake. In addition, the therapeutic benefit of radioiodide treatment in combination with viral oncolysis and external beam radiotherapy was measured. In vitro viral cell killing of prostate cancers was dose- and time-dependent and was through apoptotic mechanisms. Importantly, combined virus therapy and iodizing radiation did not adversely affect oncolysis. NIS gene expression in infected cells was functional and mediated uptake of radioiodide both in vitro and in vivo. Therapy experiments with both xenograft and immunocompetent Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse models showed that the addition of radioiodide to VV-NIS-infected tumors was more effective than each single-agent therapy, restricting tumor growth and increasing survival. In conclusion, VV-NIS is effective in prostate cancer models. This treatment modality would be an attractive complement to existing clinical radiotherapy practice.

S-Nitrosylation of NF-κB p65 Inhibits TSH-Induced Na+/I− Symporter Expression

Nitric oxide (NO) is a ubiquitous signaling molecule involved in a wide variety of cellular physiological processes. In thyroid cells, NO-synthase III-endogenously produced NO reduces TSH-stimulated thyroid-specific gene expression, suggesting a potential autocrine role of NO in modulating thyroid function. Further studies indicate that NO induces thyroid dedifferentiation, because NO donors repress TSH-stimulated iodide (I(-)) uptake. Here, we investigated the molecular mechanism underlying the NO-inhibited Na(+)/I(-) symporter (NIS)-mediated I(-) uptake in thyroid cells. We showed that NO donors reduce I(-) uptake in a concentration-dependent manner, which correlates with decreased NIS protein expression. NO-reduced I(-) uptake results from transcriptional repression of NIS gene rather than posttranslational modifications reducing functional NIS expression at the plasma membrane. We observed that NO donors repress TSH-induced NIS gene expression by reducing the transcriptional activity of the nuclear factor-κB subunit p65. NO-promoted p65 S-nitrosylation reduces p65-mediated transactivation of the NIS promoter in response to TSH stimulation. Overall, our data are consistent with the notion that NO plays a role as an inhibitory signal to counterbalance TSH-stimulated nuclear factor-κB activation, thus modulating thyroid hormone biosynthesis.

Monitoring the migration of bone marrow derived mesenchymal stem cells to intracranial glioma by sodium iodide sympoter

Objective To construct a recombinant lentiviral expression vector containing NIS and EGFP gene, and to explore the feasibility of NIS gene for monitoring the bone marrow derived mesenchymal stem cells (BMSCs) migration to the intracranial glioma. Methods The NIS and EGFP gene fragments were subcloned into lentiviral vector pLVX-puro, then packaged and amplified in HEK293T cells to obtain recombinant lentivirus pLVX-CMV-NIS-EGFP. pLVX-CMV-0-EGFP was constructed as control. BMSCs were isolated, cultured, and transfected by lentivirus. The antibiotic-resistant transfected BMSCs (BMSCs-NIS-EGFP and BMSCs-EGFP) were selected. The expression of NIS gene was examined by Western blot. Functional NIS activity was confirmed by the uptake of 125I and the inhibition effect of NaClO4. The nude mice intracranial glioma models were established. MicroSPECT was performed at 24 h post BMSCs-NIS-EGFP injection via the tail vein. Results pLVX-CMV-NIS-EGFP and pLVX-CMV-0-EGFP were successfully constructed and packaged. BMSCs were successfully isolated and cultured. Stable cell lines BMSCs-NIS-EGFP and BMSCs-EGFP were constructed after lentivirus transfection and puromycin selection. The expression of NIS gene was detected by Western blot in BMSCs-NIS-EGFP, but not in BMSCs-EGFP. BMSCs-NIS-EGFP showed significantly more uptake of 125I(nearly 10 times than the uptake in BMSCs-EGFP) and the uptake could be significantly inhibited by NaClO4. The nude mice intracranial glioma models were successfully established and the BMSCs-NIS-EGFP in glioma foci could be visualized by microSPECT imaging at 24 h post injection. Conclusions A recombinant lentivirus containing NIS gene could be successfully constructed for monitoring BMSCs migration towards intracranial glioma. It might provide evidence on the research of BMSCs and NIS gene mediated therapy for glioma. Key words: Glioma; Bone marrow; Stem cells; Sodium/iodide symporter; Tomography, emission-computed, single-photon; Mice, nude

Molecular In Vivo Imaging Using a Noninvasive Cardiac-Specific MLC-2v Promoter Driven Dual-Gene Recombinant Lentivirus Monitoring System.

BackgroundOur study aimed to demonstrate the feasibility of using the sodium/iodide symporter (NIS) to monitor vascular endothelial growth factor (VEGF165) expression in vivo.MethodsWe constructed a recombinant lentivirus plasmid with the MLC-2v promoter driving the sodium/iodide symporter (NIS) reporter gene linked to the VEGF165 gene. Expression of NIS and VEGF gene were identified by Western blot. On days 2 and 54, 99mTc-MIBI imaging was used to evaluate changes in myocardial ischemia. Noninvasive 125I micro-SPECT/CT imaging was used to assess the expression of NIS reporter gene dynamically over the next 2 months.ResultsWestern blot analysis showed that both NIS and VEGF165 were highly expressed in rat cardiomyoblast H9C2 cells transduced with Lenti-MLC-2v-NIS--VEGF165. 125I micro-SPECT/CT reporter imaging showed higher uptake in mouse myocardium transduced with Lenti-MLC-2v-VEGF165-IRES-NIS. NIS expression peaked on day 1 after transduction followed by a progressive decline to negligible levels by day 21. On day 1, mean 125I activity value in group 1 was higher than that in group 2 (P<0.05). The mean 125I activity value in group 3 was statically lower than that in group 1 and 2 (P<0.01). On day 60, 125I uptakes in test and positive control groups became very low, and no significant differences in the mean 125I activity values were detected between group 1 and group 2 (P = 0.531 > 0.05). In group 1 (test group), 99mTc-MIBI SPECT/CT revealed improvements in perfusion and wall thickening in the apical anterior wall. Mean IOD values of NIS and CD34 were significantly higher in group 1 than group 3 (P<0.05). Our study proved mean I-125 uptake was significantly correlated with mean IOD value of NIS and CD34 (P<0.05).ConclusionThis study demonstrates the feasibility of using the NIS gene to monitor VEGF165 expression in a mouse myocardial ischemia model.

551. Longitudinal High Resolution 3-D Imaging of AAV Gene Expression In Vivo

Adeno-associated viral (AAV) are small non-integrating vectors that are being developed for a number of gene therapy applications. Different AAV serotypes, armed with constitutive or tissue specific promoters, capable of infecting various tissue types have been engineered to enable preferential transduction of the target tissues. In order to rapidly screen and evaluate the best AAV serotype and promoter for high levels of transgene expression in a specific organ, we have been developing the sodium iodide symporter (NIS) as a reporter gene for screening the biodistribution of AAV vectors. NIS is expressed endogenously in thyroid follicular cells where its role is to concentrate iodine for the synthesis of thyroid hormones. For more than 70 years, thyroidal NIS expression has been provided the basis for I-123 gamma camera or SPECT/CT imaging in thyroid disorders and for I-131 tumor ablation of metastatic thyroid cancer. Using NIS imaging, we would be able to longitudinally monitor gene expression in the same animal over time, from rodents, dogs, nonhuman primates and in human subjects. In this study, we have generated a panel of AAV vectors of serotype 1, 2, 5, 8, and 9 and administered the vectors via various routes of delivery. The mice and rats were imaged using SPECT (I-125 or Tc99m pertechnetate) isotopes on a high resolution microSPECT/CT scanner. Results indicate robust AAV gene expression by 2 weeks post vector administration. View Large Image | Download PowerPoint Slide[Figure 1: AAV mediated NIS gene expression in the liver and heart of mice post IV administration of vector]. For example, AAV9-CMV-NIS gave high levels of cardiac gene expression and interestingly, of subcutaneous brown fat (scapular region). In contrast AAV1-CMV-NIS resulted in highest gene expression in the subcutaneous brown fat only whereas AAV-LST-NIS (liver specific promoter) gave only NIS expression in the liver. When the regions of interests were quantitated, we found that AAV-NIS gene expression was maintained over 90 days in immunocompetent rats and over 200 days in immunocompromised mice. Using NIS as the reporter gene, we are able to use the same vector construct and perform similar noninvasive NIS imaging studies in large animal models using the same SPECT isotopes in combination with clinical SPECT/CT scanners. In contrast to traditional biodistribution studies utilizing PCR analysis or luciferase imaging, NIS imaging enables rapid identification of the organs supporting high levels of transgene expression.