Targeting sodium/iodide symporter gene expression for estrogen-regulated imaging and therapy in breast cancer.

Expression of the sodium iodide symporter (hNIS) has been detected in breast cancer tissue, but frequently, not at the levels necessary to mediate (131)I accumulation. Transducing the hNIS gene into breast cancer cells with adenovirus could be a tractable strategy to render breast cancer susceptible to radioiodide therapy. We constructed the replication-incompetent virus, AdSERE, in which an estrogen-responsive promoter directs the expression of hNIS. In vitro, we demonstrate that AdSERE mediates hNIS expression and iodide uptake in ER+ breast cancer cells. In vivo, we show that AdSERE-infected ER+ tumors can be imaged due to tracer accumulation; in addition, AdSERE in combination with therapeutic doses of (131)I suppresses tumor growth.

Journey of the iodide transporter NIS: from its molecular identification to its clinical role in cancer.

The Na+/I- symporter (NIS) is an intrinsic plasma membrane protein that mediates the active transport of I- in the thyroid, lactating mammary gland, stomach and salivary glands. The presence of NIS in the thyroid is exploited in diagnostic scintigraphic imaging and radioiodide therapy in thyroid cancer. The continued rapid progress in NIS research (aimed at the elucidation of the Na+-dependent I- transport mechanism, the analysis of NIS structure-function relations and the study of the tissue-specific regulation of NIS at all levels), holds potentially far-reaching medical applications beyond thyroid disease, in breast cancer and malignancies in other tissues.

Molecular analysis of the sodium/iodide symporter: impact on thyroid and extrathyroid pathophysiology.

The Na(+)/I(-) symporter (NIS) is an intrinsic membrane protein that mediates the active transport of iodide into the thyroid and other tissues, such as salivary glands, gastric mucosa, and lactating mammary gland. NIS plays key roles in thyroid pathophysiology as the route by which iodide reaches the gland for thyroid hormone biosynthesis and as a means for diagnostic scintigraphic imaging and for radioiodide therapy in hyperthyroidism and thyroid cancer. The molecular characterization of NIS started with the 1996 isolation of a cDNA encoding rat NIS and has since continued at a rapid pace. Anti-NIS antibodies have been prepared and used to study NIS topology and its secondary structure. The biogenesis and posttranslational modifications of NIS have been examined, a thorough electrophysiological analysis of NIS has been conducted, the cDNA encoding human NIS (hNIS) has been isolated, the genomic organization of hNIS has been elucidated, the regulation of NIS by thyrotropin and I(-) has been analyzed, the regulation of NIS transcription has been studied, spontaneous NIS mutations have been identified as causes of congenital iodide transport defect resulting in hypothyroidism, the roles of NIS in thyroid cancer and thyroid autoimmune disease have been examined, and the expression and regulation of NIS in extrathyroidal tissues have been investigated. In gene therapy experiments, the rat NIS gene has been transduced into various types of human cells, which then exhibited active iodide transport and became susceptible to destruction with radioiodide. The continued molecular analysis of NIS clearly holds the potential of an even greater impact on a wide spectrum of fields, ranging from structure/function of transport proteins to the diagnosis and treatment of cancer, both in the thyroid and beyond.

Molecular study of the sodium-iodide symporter (NIS): a new field in thyroidology.

The active transport of iodide into the thyroid is mediated by the Na(+)-I- symporter (NIS), an intrinsic membrane protein. NIS plays key roles in thyroid pathophysiology as the route by which I- reaches the gland for thyroid hormone biosynthesis, and as a means for diagnostic scintigraphic imaging and for radioiodide therapy in thyroid cancer. The molecular characterization of NIS started with the isolation in 1996 of a cDNA encoding rat NIS, and has subsequently led to a virtually new field in thyroidology. The research reviewed in this article clearly has far-reaching implications in the areas of structure/function of transport proteins, thyroid pathophysiology, hormone action mechanisms, cell differentiation and cancer.

N-linked glycosylation of the thyroid Na+/I- symporter (NIS). Implications for its secondary structure model.

The Na+/I- symporter (NIS), a 618-amino acid membrane glycoprotein that catalyzes the active accumulation of I- into thyroid cells, was identified and characterized at the molecular level in our laboratory (Dai, G., Levy, O., and Carrasco, N. (1996) Nature 379, 458-460). Because mature NIS is highly glycosylated, it migrates in SDS-polyacrylamide gel electrophoresis as a broad polypeptide of higher molecular mass (approximately 90-110 kDa) than nonglycosylated NIS (approximately 50 kDa). Using site-directed mutagenesis, we substituted both separately and simultaneously the asparagine residues in all three putative N-linked glycosylation consensus sequences of NIS with glutamine and assessed the effects of the mutations on function and stability of NIS in COS cells. All mutants were active and displayed 50-90% of wild-type NIS activity, including the completely nonglycosylated triple mutant. This demonstrates that to a considerable extent, function and stability of NIS are preserved in the partial or even total absence of N-linked glycosylation. We also found that Asn225 is glycosylated, thus proving that the hydrophilic loop that contains this amino acid residue faces the extracellular milieu rather than the cytosol as previously suggested. We demonstrated that the NH2 terminus faces extracellularly as well. A new secondary structure model consistent with these findings is proposed.

Identification of a structural requirement for thyroid Na+/I- symporter (NIS) function from analysis of a mutation that causes human congenital hypothyroidism.

Patients with congenital lack of I transport do not accumulate I in their thyroids, often resulting in severe hypothyroidism. A single amino acid substitution in the thyroid Na+/I- symporter (NIS), proline replacing threonine at position 354 (T354P), was recently identified as the cause of this condition in two independent patients. Here we report that the lack of I- transport activity in T354P NIS generated by site-directed mutagenesis, is not due to a structural change induced by proline, but rather to the absence of a hydroxyl group at the beta-carbon of the amino acid residue at position 354. Hence, this hydroxyl group is essential for NIS function.

The Na+/I- symporter (NIS): recent advances.

The Na+/I- symporter (NIS) catalyzes the accumulation of iodide into thyroid cells, an essential step in the biosynthesis of thyroid hormones. As a result of the isolation of the rat NIS cDNA, steadfast advances in the study of NIS at the molecular level have resulted in the following accomplishments: generation of high-affinity anti-NIS antibodies, elucidation of NIS stoichiometry and specificity by electrophysiological analysis, biochemical and immunological experimental testing of the proposed NIS secondary structure model, monitoring the regulation of NIS protein expression by thyroid stimulating hormone and iodide, characterization of the rat NIS gene promoter, isolation of the cDNA clone encoding human NIS and subsequent determination of human NIS genomic organization, description of NIS mutations in patients with congenital lack of iodide transport, and the molecular identification of NIS in extrathyroidal tissues.

Identification and quantitation of iodotyrosines and iodothyronines in proteins using high-performance liquid chromatography by photodiode-array ultraviolet-visible detection.

We describe a new method for the separation, identification and quantitation of iodotyrosines and iodothyronines [3-monoiodo-L-tyrosine (MIT), 3,5-diiodo-L-tyrosine (DIT), L-thyronine (T0), 3,5-diiodo-L-thyronine (T2), 3,5,3'-triiodo-L-thyronine (T3), reverse 3,3',5'-triiodo-L-thyronine (rT3) and 3,3',5,5'-tetraiodo-L-thyronine (T4)]. Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed on a Nucleosil C8 column with photodiode-array UV-Vis detection. A clearly defined elution profile was obtained of each iodoamino acid (iodotyrosines and iodothyronines) using a linear gradient from 20 to 80% phase B (90% acetonitrile, 10% water, 0.1% TFA), phase A (water, 0.1% TFA, pH 2.0) eluted over 40 min. Iodoamino acid composition was determined, taking into account retention times and spectral characteristics. Thyroid protein samples were digested enzymatically and the complex mixture of IAA was then injected onto the RP-HPLC system. A photodiode-array detector with a dynamic range in the UV-Vis region was used in the HPLC system to monitor the absorbance at different wavelengths continuously, collecting data which were compared with standard samples. Each IAA was quantitated using linear calibration curves obtained at 280 nm. This method allowed identification and quantitation of iodoamino acids from diverse sources in the range 2-500 ng, avoiding the need to radiolabel samples. The technique was tested with in vitro iodinated and non-iodinated human thyroglobulin and the recoveries ranged from 84 to 91%.