A recombinant measles virus (MV) expressing the sodium iodide symporter (NIS) is being considered for therapy of advanced multiple myeloma. Auger electrons selectively damage cells in which the isotope decays. We hypothesized that the Auger electron emitting isotope 125I can be used to control viral proliferation. MV was engineered to express both carcinoembryonic antigen and NIS (MV-NICE). Cells were infected with MV-NICE and exposed to 125I with appropriate controls. MV-NICE replication in vitro is inhibited by the selective uptake of 125I by cells expressing NIS. Auger electron damage is partly mediated by free radicals and abrogated by glutathione. In myeloma xenografts, control of MV-NICE with 125I was not possible under the conditions of the experiment. MV-NICE does not replicate faster in the presence of radiation. Auger electron emitting isotopes effectively stop propagation of MV vectors expressing NIS in vitro. Additional work is necessary to translate these observations in vivo.

The sodium-iodide symporter (NIS) is primarily a thyroid protein, providing for the accumulation of iodide for biosynthesis of thyroid hormones. Native NIS expression has made possible the use of radioactive iodide to image and treat thyroid disease successfully. The current study, using adult male beagle dogs, was carried out in preparation for a Phase I clinical trial of adenovirus-mediated NIS gene (approved symbol SLC5A5) therapy for prostate cancer. Direct intraprostatic injection of virus (Ad5/CMV/NS) was followed by iv injection of 3 mCi 123I and serial image acquisition. The dogs were then given a therapeutic dose of 131I (116 mCi/m2) and observed for 7 days. SPECT/CT fusion imaging revealed clear images of the NIS-transduced prostates. Dosimetry calculations revealed an average absorbed dose to the prostate of 23 +/- 42 cGy/mCi 131I, with acceptably low radiation doses to other organs. This study demonstrated the successful introduction of localized NIS expression in the prostate gland of dogs, with no vector-related toxicity observed. None of the animals experienced any surgical complications, and serum chemistry panels showed no significant change following therapy. The results presented provide further evidence of the safety and efficacy of NIS as a therapeutic gene and support translation of this work into the clinical setting.

Purpose: Expression of the sodium iodide symporter (NIS) in the thyroid gland provides for effective imaging and treatment of thyroid cancer using radiolabeled iodide. Transfer of NIS into other tumors would expand the utility of this treatment to tumors of nonthyroid origin. MUC1 is a transmembrane glycoprotein that is overexpressed in many tumor types, including breast, pancreatic, and ovarian. The aim of this study was to create a construct containing NIS under the control of the MUC1 promoter to target expression specifically to MUC1-positive breast cancer cells. Experimental Design: A replication-deficient adenoviral construct was created containing the MUC1 promoter followed by the human NIS gene. Iodide uptake assays, Western blot, and immunohistochemistry were used to confirm NIS expression and function. Breast cancer xenografts in mice were infected with Ad5/MUC1/NIS and then imaged and treated using radioiodide. Results: A 58-fold increase in iodide uptake was observed in infected MUC1-positive T47D cells with no significant increase observed in MUC1-negative MDA-MB-231 cells or in cells infected with the control virus. The in vivo study yielded clear images of Ad/MUC1/NIS-infected tumor xenografts using 123I. Administration of a therapeutic dose of 131I resulted in an 83% reduction in tumor volume, whereas control tumors continued to increase in size (P < 0.01). Conclusions: These results show that the MUC1 promoter is capable of directing efficient and selective expression of the NIS gene in MUC1-positive breast tumor cells. This could potentially have applications for both imaging and therapy in a range of MUC1-positive tumor types.

The thyroidal sodium iodide symporter (NIS) in combination with various radioactive isotopes has shown promise as a therapeutic gene in various tumor models. Therapy depends on adequate retention of the isotope in the tumor. We hypothesized that in the absence of iodide organification, isotope trapping is a dynamic process either due to slow efflux or re-uptake of the isotope by cells expressing NIS. Iodide efflux is slower in ARH-77 and K-562 cells expressing NIS compared to a thyroid cell line. Isotope retention half times varied linearly with the number of cells expressing NIS. With sufficient NIS expression, iodide efflux is a zero-order process. Efflux kinetics in the presence or absence of perchlorate also supports the hypothesis that iodide re-uptake occurs and contributes to the retention of the isotope in tumor cells. Iodide organification was insignificant. In vivo studies in tumors composed of mixed cell populations confirmed these observations.

We reported recently the induction of androgen-dependent iodide uptake activity in the human prostatic adenocarcinoma cell line LNCaP using a prostate-specific antigen (PSA) promoter-directed expression of the sodium iodide symporter (NIS) gene. This offers the potential to treat prostate cancer with radioiodine. In the current study, we examined the regulation of PSA promoter-directed NIS expression and therapeutic effectiveness of (131)I in LNCaP cells by all-trans-retinoic acid (atRA). For this purpose, NIS mRNA and protein expression levels in the NIS-transfected LNCaP cell line NP-1 were examined by Northern and Western blot analysis following incubation with atRA (10 (-9) to 10(-6) M) in the presence of 10(-9) M mibolerone (mib). In addition, NIS functional activity was measured by iodide uptake assay, and in vitro cytotoxicity of (131)I was examined by in vitro clonogenic assay. Following incubation with atRA, NIS mRNA levels in NP-1 cells were stimulated 3-fold in a concentration-dependent manner, whereas NIS protein levels increased 2.3-fold and iodide accumulation was stimulated 1.45-fold. This stimulatory effect of atRA, which has been shown to be retinoic acid receptor mediated, was completely blocked by the pure androgen receptor antagonist casodex (10(-6) M), indicating that it is androgen receptor dependent. The selective killing effect of (131)I in NP-1 cells was 50% in NP-1 cells incubated with 10(-9) M mib. This was increased to 90% in NP-1 cells treated with atRA (10(-7) M) plus 10(-9) M mib. In conclusion, treatment with atRA increases NIS expression levels and selective killing effect of (131)I in prostate cancer cells stably expressing NIS under the control of the PSA promoter. Therefore atRA may be used to enhance the therapeutic response to radioiodine in prostate cancer cells following PSA promoter-directed NIS gene delivery.

Prostate cancer is one of the most promising candidates for sodium iodide symporter (NIS)-mediated gene therapy. Adenovirus-mediated expression of NIS that is driven by prostate-specific promoters induces generous radioiodine accumulation in prostate cancer cells that may be used for therapy with (131)I. We have recently developed a replication-deficient adenovirus carrying the human NIS cDNA linked to a composite probasin promoter, ARR(2)PB, aiming toward specific expression of the human NIS gene (h-NIS) in prostate tissue for targeted radioactive iodide therapy of prostate cancer (Ad-ARR(2)PB/hNIS). The ability of Ad-ARR(2)PB/hNIS to cause NIS expression in tumor cells was characterized by iodide uptake assay and compared with Ad-CMV/hNIS in which the h-NIS expression is driven by the cytomegalovirus (CMV) promoter. Androgen-dependent prostate cancer cell lines (LNCaP) and non-prostate origin tumor cell lines (SNU449, MCF-7, HCT116, OVCAR-3, and Panc-1) were infected with the viral constructs, and perchlorate-sensitive (125)I uptake and NIS protein expression were measured. Ad-ARR(2)PB/hNIS-infected LNCaP cells showed androgen-dependent and perchlorate-sensitive iodide uptake. Iodide accumulation in LNCaP cells infected with Ad-ARR(2)PB/hNIS, followed by incubation with synthetic androgen, was 5.3-fold increased compared with those coincubated with perchlorate (15,184 +/- 1,173 cpm versus 2,837 +/- 187 cpm). Ad-ARR(2)PB/hNIS-infected LNCaP cells revealed a 3.2-fold increase of iodide accumulation compared with those infected with Ad-CMV/hNIS (multiplicity of infection = 30). Iodide uptake in a panel of non-prostate tumor cell lines infected with Ad-ARR(2)PB/hNIS was no more than 2,500 cpm, demonstrating the tissue specificity of this construct. These results indicate that Ad-ARR(2)PB/hNIS can be used to achieve high-magnitude and tissue-specific expression of h-NIS in prostate tissue and is a promising candidate for cancer gene therapy of prostate cancer.

Multiple myeloma is a disseminated neoplasm of terminally differentiated plasma cells that is incurable with currently available therapies. Although the disease is radiosensitive, external beam radiation leads to significant toxicity due to sensitive end-organ damage. Thus, genetic approaches for therapy are required. We hypothesized that the incorporation of immunoglobulin promoter and enhancer elements in a self-inactivating (SIN) lentiviral vector should lead to specific and high-level transgene expression in myeloma cells. A SIN lentivector with enhanced green fluorescent protein (EGFP) expression under the control of a minimal immunoglobulin promoter as well as the Kappa light chain intronic and 3' enhancers transduced myeloma cell lines with high efficiency (30%-90%). EGFP was expressed at a high level in myeloma cells but silent in all nonmyeloma cell lines tested compared with the cytomegalovirus (CMV) promoter/enhancer. Transduction of myeloma cells with the targeted vector coding for the human sodiumiodide symporter (hNIS) led to hNIS expression by these cells allowing them to concentrate radioiodine up to 18-fold compared with controls. Tumor xenografts in severe combined immunodeficiency mice expressing hNIS could be imaged using iodine-123 (123I) and shown to retain iodide for up to 48 hours. These tumor xenografts were completely eradicated by a single dose of the therapeutic isotope iodine-131 (131I) without evidence of recurrence up to 5 months after therapy. We conclude that lentivectors can be transcriptionally targeted for myeloma cells and the use of hNIS as a therapeutic gene for myeloma in combination with 131I needs further exploration.

The iodide-concentrating ability of the thyroid gland is essential to the production of thyroid hormone. We report the nucleotide and amino acid sequence of the mouse sodium iodide symporter (mNIS), which mediates this activity within the thyroid gland. An open reading frame of 1,857 nucleotides codes for a protein of 618 amino acids with 95% identity to rat NIS and 84% identity to human NIS. Transient expression of the mNIS cDNA in Chinese hamster ovary (CHO) cells, a nonthyroid cell line, resulted in sodium-dependent, perchlorate-sensitive iodide uptake. Western blot analysis of membrane preparations of CHO cells transiently transfected with mNIS cDNA showed a band of 90 kd when probed with an antibody directed against rat NIS. mNIS will serve as an important reagent in determining the role of NIS in experimental thyroid diseases and for monitoring the immune response to in animal models of NIS-mediated gene therapy.

BACKGROUND:The human sodium iodide symporter (hNIS) is a transmembrane protein that mediates the active transport of iodide in the thyroid gland. Following cloning of NIS, NIS expression has been detected in a broad range of nonthyroidal tissues, suggesting that iodide transport in these tissues is conferred by the expression of functional NIS protein.METHODS:The aim of this study was to examine functional hNIS expression in kidney by reverse transcription-polymerase chain reaction (RT-PCR), ribonuclease protection assay (RPA), immunohistochemistry, and Western blot analysis accompanied by iodide accumulation studies in kidney cells.RESULTS:Using a pair of full-length hNIS-specific oligonucleotide primers, RT-PCR followed by Southern hybridization revealed hNIS mRNA expression in normal human kidney tissue. The PCR products were subjected to automated sequencing and revealed full identity with the published human thyroid-derived NIS cDNA sequence. Furthermore, positive protected bands indicating the presence of hNIS mRNA were apparent in RPA gel lanes corresponding to human kidney cells as well as Chinese hamster ovary (CHO) cells stably transfected with hNIS cDNA and Graves' thyroid tissue. Immunohistochemical analysis of normal human kidney tissue using a mouse monoclonal hNIS-specific antibody showed marked hNIS-specific immunoreactivity confined to tubular cells, while no hNIS-specific immunoreactivity was detected in the glomeruli. NIS protein expression in human kidney cells was further confirmed by Western blot analysis. In addition, accumulation of (125)I was detected in human kidney cells in vitro and was shown to be sodium dependent and sensitive to perchlorate.CONCLUSIONS:Functional hNIS expression was demonstrated in the renal tubular system, suggesting that renal iodide transport may be, at least in part, an active process driven by NIS.

Radioiodine therapy, the most effective form of systemic radiotherapy available, is currently useful only for thyroid cancer because of thyroid-specific expression of the sodium iodide symporter (NIS). Here we explore the efficacy of a novel form of gene therapy using adenovirus-mediated in vivo NIS gene transfer followed by 131I administration for treatment of prostate cancer. Prostate cancer xenografts in nude mice injected with an adenovirus carrying the NIS gene linked to the cytomegalovirus (CMV) promoter revealed highly active uptake of radioiodine. Following administration of 3 mCi of 131I, we observed an average tumor volume reduction of 84 ± 12%. These results show for the first time that in vivo NIS gene delivery into non-thyroidal tumors is capable of inducing accumulation of therapeutically effective radioiodine doses and might therefore represent an effective and potentially curative therapy for prostate cancer.

The ability of thyroid cancers to concentrate radioiodine (RAI) is dependent, in part, upon the expression and functional integrity of the sodium iodide symporter (NIS). However, some differentiated thyroid carcinomas (DTCs) and most undifferentiated thyroid carcinomas lack the ability to concentrate iodide and are thereby insensitive to 131I therapy. Variation of NIS protein expression may be an important factor in this behavior. We wished to determine whether NIS protein expression in primary DTC tumors correlated with the subsequent RAI uptake by metastatic lesions in the same patients. We obtained paraffin-embedded tissue specimens from 60 patients with metastatic thyroid cancer who had undergone total or near-total thyroidectomy at the Mayo Clinic for DTC and had known presence or absence of RAI uptake in their tumor deposits determined by total body scanning after thyroid hormone withdrawal. Tissue sections from the primary intrathyroidal tumors were subjected to immunostaining (IS) using a monoclonal antibody against human NIS. Slides were subsequently examined for specific IS by two independent reviewers. For each patient, whole body scan (WBS) uptake was recorded, and correlation between results of IS and WBS was analyzed. Of 43 patients with a positive WBS, 37 also had positive IS of their tumors. In six patients with negative IS, a positive WBS was documented, and in three of these cases TSH at the time of surgery was less than 0.3 mIU/liter. Of the 17 patients with negative WBS, 10 were also negative on IS. Positive IS accurately predicted a positive scan in our study in 84% of cases; the ability of the IS to detect all cases with a positive scan was 86%, and it increased to 90% when patients who were receiving thyroid hormone therapy at the time of surgery were excluded from the analysis. Overall, the results of our retrospective study suggest that NIS IS of the thyroidal primary tumor in patients with papillary and follicular thyroid cancers has substantial ability to predict the behavior of subsequent deposits of metastatic and recurrent cancer with respect to iodine trapping and concentration. Our findings require confirmation in prospective studies to more accurately determine the predictive ability of the test and its role in the postoperative management of patients with DTC. If confirmed, NIS IS of DTC primary lesions may prove useful in the management of patients with known or suspected metastatic thyroid cancer.

Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) by targeted NIS gene transfer might offer the possibility of radioiodine therapy of prostate cancer. Therefore, we investigated radioiodine accumulation and therapeutic effectiveness of 131I in NIS-transfected prostate cancer cells in vitro and in vivo. The human prostatic adenocarcinoma cell line LNCaP was stably transfected with NIS cDNA under the control of the prostate-specific antigen promoter. The stably transfected LNCaP cell line NP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in vitro that resulted in selective killing of these cells by 131I in an in vitro clonogenic assay. Xenografts were established in athymic nude mice and imaged using a gamma camera after i.p. injection of 500 microCi of 123I. In contrast to the NIS-negative control tumors (P-1) which showed no in vivo uptake of 123I, NP-1 tumors accumulated 25-30% of the total 123I administered with a biological half-life of 45 h. In addition, NIS protein expression in LNCaP cell xenografts was confirmed by Western blot analysis and immunohistochemistry. After a single i.p. application of a therapeutic 131I dose (3 mCi), significant tumor reduction was achieved in NP-1 tumors in the therapy group compared with P-1 tumors and tumors in the control group. In conclusion, a therapeutic effect of 131I has been demonstrated in prostate cancer cells after induction of tissue-specific iodide uptake activity by prostate-specific antigen promoter-directed NIS expression in vitro and in vivo. This study demonstrates the potential of NIS as a novel therapeutic gene for nonthyroidal cancers, in particular prostate cancer.

The recent cloning of the thyroidal protein that is responsible for iodide transport, the sodium iodide symporter (hNIS), has made possible studies designed to characterize its structure, function and expression in thyroidal tissues. Using a mannose binding protein (MBP)-hNIS fusion protein as antigen, we have developed mouse monoclonal antibodies against hNIS to utilize as tools in such studies. Twenty-four clones were initially recovered which recognized the MBP-hNIS fusion protein, but only two of them were specific for hNIS while the others recognized MBP alone. Both antibodies were found to be immunoglobulin G (IgG) 1kappa (kappa). The specificity of antibodies was tested by Western blotting using membranes prepared from COS-7 cells transiently transfected with the pcDNA3 plasmid containing the full-length hNIS cDNA, or cells transfected with the pcDNA3 vector. A major band with a molecular weight (MW) of approximately 97 kDa, and several minor bands with MW of approximately 160 kDa, approximately 68 kDa, approximately 30 kDa and approximately 15 kDa, were detected specifically in the hNIS-transfected cells. After enzymatic deglycosylation, the major band was present at 68 kDa, as expected based upon the amino acid sequence of hNIS. Immunohistochemistry was performed with several different types of thyroid tissue and non-thyroidal tissues, using the monoclonal antibodies. Strong immunostaining was observed in Graves' tissue, with intermediate staining in papillary and follicular thyroid cancers and an absence of staining in Hürthle cell cancer. The staining was specific for the follicular epithelium and was concentrated in the basolateral portion of the cell membrane. These monoclonal hNIS antibodies should prove useful in the characterization of NIS expression in benign and malignant thyroid tissue and in studies characterizing its structure and function.

The thyroid sodium-iodide symporter (NIS) is responsible for iodide concentrating ability within thyroid follicular cells. We sought to develop monoclonal antibodies against human NIS (hNIS) for use as reagents in structure-function studies of the protein, as well as potential tools in the assessment of NIS expression in benign and malignant thyroid tissues. Synthetic peptides corresponding to the second ExMD and to the carboxy-terminal ExMD of hNIS were produced and utilized as antigens to develop monoclonal antibodies, which were tested by Western blotting using membranes prepared from COS-7 cells transiently transfected with a pcDNA3 plasmid containing the gene for the full-length hNIS, or a control vector. Western blotting showed a major band with molecular weight (MW) of approximately 97 kDa and several minor bands with MW of approximately 160 kDa, 68 kDa, 30 kDa, and 15 kDa, all specific for hNIS-transfected cells. Immunohistochemistry was performed in various types of thyroid tissues and nonthyroidal tissues, using the monoclonal antibodies. Strong immunostaining was observed in Graves' tissue, intermediate staining in papillary and follicular thyroid cancer, and no staining in Hürthle cell cancer or in nonthyroidal tissue. The staining was specific for the follicular epithelium in each of the tissues and was most intense in the basolateral portion of the cell membrane. Overall, our observations indicate that the monoclonal antibodies are specific for hNIS and will be invaluable reagents for investigating the role of NIS in thyroid disease.

Toxic multinodular goiter (TMNG) represents a frequent cause of endogenous hyperthyroidism, affecting 5-15% of such patients (with higher frequencies reported in iodine-deficient areas of the world). Although mutations of human TSH receptor (hTSHR) have been described in autonomously functioning thyroid nodules (AFTN), the role of such mutations in the pathogenesis of TMNG remains unclear. To search for alterations of hTSHR in AFTN and TMNG, we performed bidirectional, dye primer automated fluorescent DNA sequencing of the entire transmembrane domain and cytoplasmic tail of hTSHR (TMD+CT-hTSHR) using DNA extracted from nodular regions of 24 patients with TMNG and 7 patients with AFTN. Eight of the 24 patients (33.3%) showed heterozygote polymorphism of codon 727 on the cytoplasmic tail of hTSHR with an amino acid substitution of aspartic acid to glutamic acid. Three of 24 (12.5%) patients with TMNG were found to carry a heterozygote mutation of codon 703, resulting in substitution of alanine with glycine. One patient had multiple heterozygote mutations including I606M (Ile to Met), A703G (Ala to Gly), Q720E (Gln to Glu), and D727E (Asp to Glu). Two patients exhibited silent polymorphism of codons 460 and 618. We found no mutation of the TMD+CT-hTSHR in 7 patients with AFTN, except for a silent polymorphism of codon 460 in 1. DNA fingerprinting of codon 727 using restriction enzyme NlaIII and genomic DNA confirmed the sequencing results in all cases, indicating that the sequence alterations were not somatic in nature. This technique was also used to examine peripheral blood genomic DNA from 52 normal individuals and 49 patients with Graves' disease; 33.3% of TMNG (P = 0.019 vs. normal subjects), 16.3% of Graves' disease patients (P = 0.10 vs. normal subjects), and 9.6% of normal individuals were heterozygous for the D727E polymorphism. Expression of the D727E hTSHR variant in eukaryotic cells (COS-7) resulted in an exaggerated cAMP response to TSH stimulation compared to that of the wild-type hTSHR. These findings indicate that a germline polymorphism of codon D727E of hTSHR is associated with TMNG, suggesting that its presence is an important predisposing genetic factor in the pathogenesis of TMNG.