Iodine [or iodide (I-) in its ionized form] is an essential nutrient, primarily because of the role it plays as an in dispensable component of the two thyroid hormones triiodothyronine (T3) and thyroxine (T4). The thyroid hormones are iodothyronines, i.e., the result of two coupled iodotyrosines, and are the only iodine-containing hormones in vertebrates. Without iodine there is no biosynthesis of thyroid hormones. Therefore, thyroid function ultimately depends on an adequate supply of iodine. A remarkably efficient and specialized system has evolved in the thyroid that ensures that most of the ingested dietary I- (the only source of I-) is accumulated in the gland and thus made available for T3 and T4 biosynthesis. The importance of this becomes more apparent when one considers that I- is rather scarce in the environment. Endemic goiter and cretinism caused primarily by insufficient dietary supply of I- remain a major health problem in many parts of the world, affecting millions of people (1). This situation dramatizes the health value of I- as a nutrient and the consequences to society of its environmental scarcity.
Iodine was discovered in 1811 by Courtois, who isolated it by treating seaweed ash with sulphuric acid (2). The ability of the thyroid to concentrate I- was first reported as early as the 19th century (3,4). The thyroid gland was found to be capable of concentrating I- by a factor of 20 to 40 with respect to its concentration in the plasma under physiological conditions. Hence the existence of a thyroid I- transpoter was inferred, and some of its properties were elucidated over the years [see (5,6,7) for reviews]. Briefly, I- accumulation in the thyroid has long been shown to be an active transport process that occurs against the I- electrochemical gradient, stimulated by thyrotropin (TSH), and blocked by the well-known “classic” competitive inhibitors, the anions thiocyanate and perchlorate. Eventually, it was determined that the thyroid I- transporter is a sodium-iodide (Na+/I-) symporter (NIS) (8,9,10,11,12), i.e., an intrinsic plasma membrane transport protein that couples the inward “downhill” translocation of Na+ with the inward “uphill” translocation of I- (Fig. 4A.1). The driving force for the process is the inwardly directed Na+ gradient generated by the Na+/K+- adenosine triphosphate (ATP)-ase (Fig. 4A.1). The ability of the thyroid to accumulate I- via NIS provided the basis for diagnostic scintigraphic imaging of the thyroid with radioiodide and has served as an effective means for pharmacologic doses of radioiodide to target and destroy hyperfunctioning thyroid tissue, such as in Graves' disease, or I- -transporting thyroid cancer cells. Therefore, the study of NIS is of great relevance to thyroid pathophysiology. Nevertheless, no molecular information on NIS was available until 1996, when after a decades-long search by numerous investigators, the DNA encoding rat NIS was finally isolated by expression cloning in Xenopus laevis oocytes (13). This development marked the beginning of the molecular characterization of NIS.
FIGURE 4A.1. Iodide(I-) transport in thyroid follicular cells. The basolateral surface of the cell is shown on the left side of the figure and the apical surface on the right. Circle: active accumulation of I-, mediated by the sodium-iodide symporter; diamond: Na+/K+- adenosine triphosphatase; rectangle: I- efflux into the colloid, mediated by apical iodide transporter, pendrin, or Cl- channels.
NIS mediates the first and key step in the process of supplying I- to the gland for thyroid hormone biosynthesis, that is, the active transport of I- against its electrochemical gradient across the basolateral plasma membrane into the cytoplasm of the follicular cells (I- uptake) (Fig. 4A.1). I- is then translocated across the apical membrane into the colloid, located in the follicular lumen (I- efflux). I- efflux into the follicular lumen has been suggested to be mediated by a different plasma membrane transporter putatively located on the apical side of the follicular cells, facing the colloid. The identity of this transporter has yet to be unequivocally established. Two different proteins, pendrin (14) and the apical iodide transporter (AIT) (15), have been proposed to play the role of mediator of I- efflux. However, it is also possible that I- efflux is mediated by Cl- channels or transporters (Fig. 4A.1).
This chapter focuses on the most recent NIS research at the molecular level, which has become an exciting new field in thyroidology, and on the impact this research is having on our understanding of thyroidal and extrathyroidal physiology and disease.
POSSIBLE ROLE OF SODIUM-IODIDE SYMPORTER IN HUMAN EVOLUTION
In a fascinating and provocative report, Dobson (16) suggested that the geographical distribution of the environmental supply of iodine and hominid adaptations to it may have played a major role in human evolution. Dobson suggested that the anatomical characteristics of Neanderthals were remarkably similar to those of modern humans who suffer from cretinism as a result of iodine deficiency. He argues persuasively that these observations support the notion that a single genetic alteration, which resulted in a better ability to trap I- in the thyroid, may explain the differences between Neanderthals and modern humans, and may account for the eventual replacement of Neanderthal populations by modern humans. In light of Dobson's article, it seems conceivable that the development of numerous characteristic and essential traits of modern humans, most notably brain development and intelligence, may have resulted in part from improved thyroidal I- transport. Although it remains to be demonstrated, if such a single genetic alteration indeed occurred, and had the described effect, it could have involved, precisely, the gene that encodes NIS.
MOLECULAR CHARACTERIZATION OF SODIUM-IODIDE SYMPORTER
Identification of the Sodium-Iodide Symporter DNA
A promising development in the search for the NIS molecule was the expression of perchlorate-sensitive Na+/I- symport activity in X. laevis oocytes by microinjection of poly A+ RNA isolated from FRTL-5 cells, a highly functional line of rat thyroid-derived cells (11). A sevenfold increase of I- accumulation over background was observed 6 to 7 days after injection. Poly A+ RNA was subsequently fractionated by sucrose gradient centrifugation, and fractions were assayed for their ability to induce I- accumulation in oocytes. The poly A+ RNA encoding NIS was found in fractions containing messenger RNA that were 2.8 to 4.0 kb in length (11). Thus, the oocyte system was shown to be of potential value for the possible expression cloning of the cDNA that encodes NIS in the absence of oligonucleotides, based on protein sequence data or anti-NIS antibodies. After a few years, this cloning strategy was successful.
Dai et al (13) generated several cDNA libraries from poly A+ RNA of FRTL-5 cells and subjected them to expression screening in X. laevis oocytes. The library was size-fractionated, and the fraction containing inserts from 2.5 to 4.5 kb was screened. The poly A+ RNA encoding NIS was found in a fraction containing messages of 2.8 to 4.0 kb in length (11). The expression cloning of NIS was carried out by measuring perchlorate-sensitive Na+/I- symport activity in oocytes microinjected with cRNAs made in vitro from pools containing decreasing numbers of cDNA clones. The cloning of NIS marked the beginning of its molecular characterization, as discussed later in the chapter.
Primary Sequence and Secondary Structure Model of Sodium-Iodide Symporter
The complete nucleotide sequence of the cloned NIS cDNA and the deduced amino-acid sequence are presented in the Dai et al study (13). Beginning with Met at position 1, a long, open-reading frame codes for a protein of 618 amino acids (relative molecular mass, 65,196). The hydropathic profile and initial secondary structure predictions of the protein led Dai et al (13,17) to suggest an intrinsic membrane protein with 12 putative transmembrane segments. However, this model has subsequently been revised, as detailed later in the chapter. The NH2 terminus was originally placed on the cytoplasmic side, given the absence of a signal sequence. The COOH terminus, which was also predicted to be on the cytoplasmic side, was found to contain a large hydrophilic region of ~70 amino acids. Levy et al demonstrated experimentally the intracellular orientation of the COOH terminus by showing that an anti-COOH terminus antibody to its epitope only after cell permeabilization (18). Three potential Asn-glycosylation sites were identified in the deduced amino acid sequence at positions 225, 485, and 497. The first was located in a predicted intracellular hydrophilic loop, while the last two were located in the last hydrophilic loop, a segment predicted to be on the extracellular face of the membrane.
Levy et al (19) obtained conclusive evidence that, contrary to previous suggestions (20), neither partial nor total lack of N-linked glycosylation impairs the activity, stability, or targeting of NIS. Using site-directed mutagenesis, Levy et al (19) substituted both separately and simultaneously the Asn residues (amino acids 225, 485, and 497) with Gln in all three putative N-linked glycosylation consensus sequences of NIS and assessed the effects of the mutations. All mutants were active and displayed 50% to 100% of wild-type NIS activity, including the completely nonglycosylated triple mutant. The half-life of non glyco sylated NIS was similar to wild-type NIS, and the Km value for I- (~20 to 30 mM) in non glycosylated NIS was virtually identical to that of wild-type NIS. These findings demonstrate that, to a considerable extent, function, targeting, and stability of NIS are present even in the total absence of N-linked glycosylation (19). Therefore, a bacterial expression system in which no N-linked glycosylation occurs might be used to overproduce NIS for structural studies.
Levy et al (19) also demonstrated that the putative N-linked glycosylation site at N225, which had originally been predicted to face intracellularly, is indeed glycosylated. Therefore, it is now clear that the hydrophilic loop that contains this sequence faces the extracellular milieu rather than the cytosol. They have proposed a 13-transmembrane-segment model to be the most likely secondary structure for NIS (Fig. 4A.2). In contrast to the original model, in which the NH2 terminus was predicted to face the cytosol on account of the lack of a signal sequence in NIS, in the current model both the NH2 terminus and the hydrophilic loop containing N225 are predicted to be on the extracellular side, and the COOH terminus faces the cytosol.
FIGURE 4A.2. Model of the secondary structure of the human sodium-iodide symporter. Iodide transport defect-causing mutations are shown in the rounded rectangles, which contain the WT amino acid letter, its position, and the letter of the amino acid causing the mutation. X, stop codon; fS, frame shift; δ, deletion.
Levy et al (19) subsequently demonstrated unequivocally that the NH2 terminus faces the external milieu, as proposed in the current model. This conclusion was reached using two independent experimental approaches. First, these authors introduced a FLAG epitope at the NH2 terminus. COS cells transfected with FLAG-containing NIS displayed I- uptake undistinguishable from that of COS cells transfected with wild-type NIS. Immuno fluorescence experiments demonstrated positive immuno reactivity with anti-FLAG antiboides (Ab) in nonpermeabilized COS cells transfected with FLAG-containing NIS. Positive immunoreactivity in nonpermeabilized cells indicates that the NH2 terminus faces externally. In contrast, immunoreactivity using anti-COOH Ab requires permeabilization because the COOH terminus faces the cytosol (18,20). The second technique took advantage of a pre vious observation that nonglycosylated NIS is active. The N-linked glycosylation amino acid sequence NNSS was introduced into the NH2 terminus of unglycosylated NIS (21). They observed glycosylation of NIS at the NH2 terminus upon transfection of NNSS-containing NIS into COS cells, thus proving that the NH2 terminus faces the lumen of the endoplasmic reticulum during biosynthesis and therefore faces the external milieu upon reaching the plasma membrane.
Sodium-Iodide Symporter Mechanism and Stoichiometry
Eskandari et al (22) have examined the mechanism, stoichiometry, and specificity of NIS by means of electrophysiological, tracer-uptake, and electron microscopic methods in X. laevis oocytes expressing NIS. These authors obtained electrophysiological recordings using the two microelectrode voltage clamp technique. They showed that an inward steady-state current (i.e., a net influx of positive charge) is generated in NIS-expressing oocytes upon addition of I- to the bathing medium, leading to depolarization of the membrane. Since the recorded current is attributable to NIS activity, this observation confirms that NIS activity is electrogenic. Simultaneous measurements of tracer fluxes and currents revealed that two Na+ ions are transported with one anion, demonstrating unequivocally a 2:1 Na+/I- stoichiometry. Therefore, the observed inward steady-state current is due to a net influx of Na+ ions.
Eskandari et al (22) determined that the turnover rate of NIS is ~36 s-1, and reported that expression of NIS in oocytes led to a ~2.5-fold increase in the density of plasma membrane protoplasmic face intramembrane particles, as ascertained by freeze-fracture electron microscopy. This is the first direct electron microscopy visualization of osten sible NIS molecules present in the oocyte plasma membrane. Moreover, on the basis of their kinetic results, these authors proposed an ordered simultaneous transport mechanism in which Na+ binds to NIS before I-, where transport of both ions is simultaneous, binding is ordered and sequential.
Specificity of Sodium-Iodide Symporter
Similar steady-state inward currents were generated by a wide variety of anions in addition to I- (including ClO3-, SCN-, SeCN-, NO-3, Br-, IO4-, and BrO3-), indicating that these anions are also transported by NIS. However, perchlorate (ClO4-), the most widely characterized inhibitor of thyroidal I- uptake, was found surprisingly to not generate a current, strongly suggesting that it is not transported (22). Yoshida et al (23) have reported, similarly, that perchlorate did not induce an inward current in Chinese hamster ovary (CHO) cells stably expressing NIS, as measured using the whole-cell patch-clamp technique. The most likely interpretation of these observations is that perchlorate is not transported by NIS, although the unlikely possibility that perchlorate is translocated by NIS on a 1:1 Na+/ClO4- stoichiometry cannot be ruled out. To unequivocally elucidate whether perchlorate is translocated or not by NIS, flux experiments would need to be performed with perchlorate labeled with 36Cl. However, the low specific activity and exorbitant cost of 36Cl make such experiments practically impossible. Yoshida et al (24) thereafter showed that perchlorate elicits no change in the membrane current in the highly functional rat thyroid cell line FRTL-5, as revealed by the whole-cell patch-clamp technique, thus strongly suggesting that perchlorate is not transported into FRLT-5 cells and supporting both their previous observations in CHO cells (23) and Eskandari et al's (22) results in oocytes. Therefore, currently available data indicate that perchlorate is a potent inhibitor of NIS that acts as a blocker, not a substrate.
REGULATION OF THE SODIUM-IODIDE SYMPORTER
Regulation of Sodium-Iodide Symporter-Transcription
Three different transcription factors have been implicated in thyroid-specific gene transcription (25,26): (a) thyroid transcription factor-1 (TTF-1), a homeodomain (HD)-containing protein present in the developing thyroid, lung, forebrain, and pituitary, as well as in the adult thyroid and lung; (b) TTF-2, a fork head protein detected in developing thyroid, anterior pituitary, and adult thyroid; and (c) Pax-8, a nuclear protein member of the murine family of paired-domain (PD)–containing genes, present in the developing thyroid, kidney, and midbrain boundary, as well as in the adult thyroid and kidney. Specific combinations of these factors have been proposed to regulate transcription of the thyroid-specific proteins thyroglobulin (Tg), thyroid peroxidase (TPO), and thyrotropin receptor (TSHR).
Genomic Organization of the Human Sodium-Iodide Symporter
The cDNA-encoding human NIS (hNIS) was identified on the expectation that hNIS would be highly homologous to rat NIS (rNIS). Using primers to the rNIS cDNA sequence (specifically rNIS nucleotide sequences 362 to 480 and 671 to 900), Smanik et al (27) amplified a cDNA fragment of hNIS from human papillary thyroid carcinoma tissue by PCR, utilized this cDNA fragment to screen a human thyroid cDNA library, and isolated a single cDNA clone encoding hNIS. The nucleotide sequence of hNIS revealed an open reading frame of 1929 nucleotides, which encodes a protein of 643 amino acids. Human NIS exhibits 84% identity and 93% similarity to rNIS, and it differs from rNIS only in that it has a 5 amino acid insertion between the last two hydrophobic domains (amino acids 485 to 488 and 499) and a 20 amino acid insertion in the COOH terminus (amino acids 618 to 637). Subsequently, Smanik et al (28) examined the expression, exon-intron organization, and chromosome mapping of hNIS. Fifteen exons encoding hNIS were found to be interrupted by 14 introns (Fig. 4A.3), and the hNIS gene was mapped to chromosome 19p. The human NIS promoter has been sequenced by three different groups (29,30,31).
FIGURE 4A.3. Correlation of the structural organization of the human sodium-iodide symporter (hNIS) gene with the NIS protein. Exons in the hNIS gene are represented by gray rectangles. The putative 13 transmembrane segments in the protein are represented by roman numerals. Exons are connected to the cor responding amino acid region of the protein by dotted lines. Localization of the iodide transport defect mutations is indicated under the schematic representation of the hNIS protein.
Analysis of the Rat and Human Sodium-Iodide Symporter Promoters
It has long been established that TSH stimulates NIS activity via the cyclic adenosine monophosphate (cAMP) pathway (20,32,33) and, more recently, that it up-regulates NIS messenger RNA (mRNA) levels (34). However, to fully understand these mechanisms, it is necessary to study the transcriptional regulation of NIS. As for rNIS, Endo et al (35) localized a TTF-1 binding site between -245 and -230 bp, in the proximal rNIS promoter (2 kb), that confers thyroid-specific transcription, but only exerts a modest effect. The same group (36) subsequently identified, in the 5′-flaking region between -1968 and +1 bp, a novel TSH-responsive element (TRE) between -420 and -385 bp upstream of the TTF-1 site in the rNIS promoter that up-regulated two- to threefold NIS expression. The TSH effect is cyclic AMP-mediated and thyroid-specific. The pro tein that binds this site is different from TTF-1, TTF-2, Pax-8, or other known transcription factors. The authors named the putative binding protein in the TRE site NTF-1 (NIS TSH-responsive factor-1). However, as the TSH up-regulation of NIS via this TRE site is lower than the regulation that the same group reported previously (~sixfold) (34), they have suggested that other transcription-binding sequences may be present upstream from the region they studied.
A thorough characterization of the upstream enhancer of the rNIS gene was reported by Ohno et al (37). These authors showed that the rNIS regulatory region contains a non-thyroid-specific promoter between -564 and -2 bp, and an enhancer located between -2264 and -2495 bp that re capitulates the most relevant aspects of NIS regulation (Fig. 4A.4). This rNIS enhancer mediates thyroid-specific gene expression by the interaction of Pax-8 with a novel cyclic AMP-dependent pathway. The NIS upstream enhancer (NUE) stimulates transcription in a thyroid-specific and cyclic AMP-dependent manner. NUE contain: two Pax-8–binding sites (PA and PB); two TTF-1 binding sites (TA and TB), which have no effect on rNIS transcription; and a degenerate cyclic AMP-responsive element (CRE) sequence (5′-TGACGCA-3′), which is important for NUE transcriptional activity (Fig. 4A.4). Interestingly, the same degenerate CRE sequence has been implicated in tissue-specific cyclic AMP response of other promoters, such as the β-hydroxylase (38), prohormone convertase 1 (39), and proenkeph alin genes (40). In NUE, both Pax-8 and the unidentified CRE-like binding factor act synergistically to obtain full TSH/cyclic AMP-dependent transcription. However, this enhancer is also able to mediate cyclic AMP-dependent transcription by a novel protein kinase A (PKA)-independent mechanism (37).
FIGURE 4A.4. Diagram of the sodium-iodide symporter (NIS) promoter indicating the major transcription start site (+1), the TATA box, the proximal promoter, and the NIS upstream enhancer (NUE). The rat proximal promoter contains a thyroid transcription factor-1 (TTF-1) binding site and a thyrotropin (TSH)-responsive element where a putative transcription factor NIS TSH-responsive factor-1 interacts. The rat NIS upstream enhancer (NUE) (top) contains two Pax-8 binding sites and a degenerative cyclic AMP-response-element sequence (CRE); and the human NUE (bottom) contains one Pax-8 binding site, one TTF-1 binding site, and one CRE-like binding site, which are important for full TSH/cyclic AMP-dependent transcription.
Transcriptional regulation of the Tg, TPO, and TSHR genes by TSH/forskolin is mediated by cyclic AMP. However, no CRE sequences have been identified for any of these, except the TSHR gene. Thus, the novel PKA-independent mechanism reported by Ohno et al (37) is highly significant, as it establishes a direct relationship between thyroid-specific transcription factors and the TSH/cyclic AMP regulation in rNIS. This picture clearly separates rNIS gene regulation from that of true thyroid-restricted genes (Tg and TPO) and also from TSHR, a notion consistent with the fact that NIS is not exclusively expressed in the thyroid gland.
Recently, a thyroid-specific, TSH-responsive, far-upstream (-9847 to -8968) enhancer in the human NIS gene—highly homologous to the rat NUE—has been reported by two groups (41,42). It contains putative Pax-8 and TTF-1 binding sites and a CRE-like sequence. The TTF-1 binding site is not required for full activity (41,42) (Fig. 4A.4).
Regulation of Sodium-Iodide Symporter in the Thyroid by Thyrotropin
TSH is the primary hormonal regulator of thyroid function overall and has long been known to stimulate thyroidal I- accumulation. Most actions of TSH take place through activation of adenylyl cyclase via the guanosine triphosphate (GTP) binding protein Gα. This cascade of events is initiated by the interaction of TSH with its receptor (i.e., TSHR) on the basolateral membrane of the follicular cells. Early observations made prior to the isolation of the NIS cDNA suggested that TSH stimulation of I- accumulation results, at least in part, from the cyclic AMP-mediated increased biosynthesis of NIS (32,33). Once the rat NIS cDNA was isolated (13) and anti-NIS Ab were generated, Levy et al demonstrated in rats that NIS protein expression is up-regulated by TSH in vivo (18). Consistent with these findings is a later observation by Uyttersprot et al (43) that the expression of NIS mRNA in dog thyroid (~3.9 kb) is dramatically up-regulated by goitrogenic treatment [i.e., pro pyl thiouracil (PTU) treatment, which leads to high TSH serum levels in vivo].
Up-regulation of thyroid NIS expression and I- uptake activity by TSH has been demonstrated not only in rats in vivo (18), but in rat thyroid-derived FRTL-5 cells (33) and human thyroid primary cultures (44,45). Marcocci et al (46), Kogai et al (34), and Ohno et al (37) have all shown that TSH up-regulates I-uptake activity by a cyclic AMP-mediated increase in NIS transcription. After TSH withdrawal, a reduction of both intracellular cyclic AMP levels and I- uptake activity is observed in FRTL-5 cells (33). This is a reversible process, as I- uptake activity can be restored either by TSH or agents that increase cyclic AMP (33,37). To investigate NIS biogenesis, Levy et al (18) carried out metabolic labeling and immunoprecipitation experiments in the presence of TSH and observed that NIS is synthesized as a precursor (~56 kDa) (19). After a 60-minute chase period, a broad, fully processed polypeptide band (~90 kDa) also became apparent.
Kaminsky et al (32) made the surprising observation that I- uptake activity is present in membrane vesicles (MV) prepared from FRTL-5 cells that, when intact, had completely lost I- uptake activity due to prolonged TSH deprivation (Fig. 4A.5). This suggests that mechanisms other than transcription may also operate to regulate NIS activity in response to TSH.
FIGURE 4A.5. Thyrotropin (TSH) regulates iodide (I-) transport and sodium-iodide symporter (NIS) expression in thyroid cells.A: I- transport activity. FRTL-5 cells were kept in the presence or absence of TSH for the indicated number of days. I- transport was measured in intact cells (dashed bars) and in membrane vesicles (MV) prepared from those cells (filled bars). I- transport measured in cells maintained in the presence of TSH and in their MV was defined as 100%. I-transport corresponding to Days 1, 3, 5, and 7 after TSH removal was expressed as the percentage of I- transport relative to Day 0. B:Schematic model to illustrate the presence of NIS activity in MV from cells deprived of TSH that, when intact, exhibit no NIS activity. This model supports the notion that active NIS molecules, initially located at the plasma membrane while TSH is present, are redistributed to intracellular compartments in response to TSH withdrawal. NIS molecules are represented as cylinders. The plasma membrane is shown in thicker lines than the intracellular compartments. C: NIS is redistributed to intracellular compartments during TSH deprivation. NIS staining was performed in FRTL-5 cells with anti-rat NIS antibodies. Cells were maintained in the presence or absence of TSH for the indicated number of days. NIS immunofluorescence in these cells was analyzed by confocal microscopy.
Kogai et al (44) have shown that TSH markedly stimulates NIS mRNA and protein levels in both monolayer and follicle-forming human primary thyrocyte cultures, whereas significant stimulation of I- uptake is observed only in follicles. These interesting observations indicate that, in addition to TSH stimulation, cell polarization and spatial organization are crucial for proper NIS activity, and suggest that NIS may be regulated by such posttranscriptional events as subcellular distribution. Riedel et al (47) have demonstrated conclusively by immunoblot analysis that NIS is present in FRTL-5 cells as late as 10 days after TSH withdrawal and that de novo NIS biosynthesis requires TSH (47). Therefore, it is clear that any NIS molecules detected in TSH-deprived FRTL-5 cells had to be synthesized prior to TSH withdrawal. This is consistent with NIS being a protein with an exceptionally long half-life, as suggested by Kogai et al (34) and Paire et al (20). Riedel et al (47) determined by pulse-chase analysis that the NIS half-life is ~5 days in the presence and ~3 days in the absence of TSH. Even though the NIS half-life in the absence of TSH is 40% shorter than in its presence, per sistence of significant I- uptake activity in MV from cells deprived of TSH (47).
Riedel et al (47) later observed that 3 days after TSH deprivation, intracellular NIS decreases at a slower rate than plasma membrane NIS, supporting the notion that active NIS molecules, initially located in the plasma membrane while TSH is present, are redistributed to intracellular compartments in response to TSH withdrawal. This model explains the presence of NIS activity in MV from cells deprived of TSH that, when intact, have no NIS activity (Fig. 4A.5). Clearly, TSH regulates I- uptake by modulating the subcellular distribution of NIS without apparently influencing the intrinsic functional status of the NIS molecules. Riedel et al (47) have also shown that NIS is phosphorylated in vivo and that serines are the main amino acid residues in which phosphorylation takes place in NIS, independently of TSH presence. However, the phosphopeptide map of NIS obtained when TSH was present was markedly different from that of when TSH was absent (47). In conclusion, TSH not only stimulates NIS transcription and biosynthesis, it is also required for targeting NIS to and/or retaining it at the plasma membrane.
Regulation of Sodium-Iodide Symporter by Iodide
The main factor regulating the accumulation of I- in the thyroid (i.e., NIS activity) other than TSH has long been considered to be I- itself. As early as 1944, Morton et al (48) reported that the biosynthesis of thyroid hormones by sheep thyroid slices was inhibited by high doses of I-. Wolff and Chaikoff reported in 1948 (49) that organic binding of I- in rat thyroid was blocked when plasma I- concentrations reached a critical high threshold, a phenomenon known as the acute Wolff-Chaikoff effect. These authors observed further that approximately 2 days later, in the presence of continued high plasma I- concentrations, an “escape,” or adaptation, from the acute effect occurred, so that the level of I- organification was restored and normal hormone biosynthesis resumed (50). The mechanism responsible for the acute Wolff-Chaikoff effect has yet to be fully elucidated, but it has been proposed to be the result of organic iodocompounds acting as mediators (51,52,53). The mechanism for the “escape” from the acute Wolff-Chaikoff effect was proposed by Braverman and Ingbar (54) to be due to a decrease in I- transport, which would presumably lead to sufficiently low intracellular I- concentrations to remove inhibition of I- organification.
The Wolff-Chaikoff effect and the ensuing “escape” constitute a highly specialized intrinsic autoregulatory system that protects the thyroid from the deleterious effects of I- overload, while at the same time ensuring adequate I- uptake for hormone biosynthesis. The level of I- capable of inhibiting I-organification and concomitantly stopping thyroid hormone synthesis is determined by the ratio of organified to nonorganified intracellular I- content, which in turn depends on the previous I- supply.
Isolation of the NIS cDNA has spurred a renewed impetus to investigate the regulatory role played by I- on NIS function. As described earlier, in vivo studies carried out by Uyttersprot et al (43) showed that I- inhibited the expression of both TPO and NIS mRNAs in dog thyroid. These observations support the proposed mechanism to explain the “escape” from the Wolff-Chaikoff effect (i.e., that it is due to a decrease in I- uptake possibly caused by down-regulation of NIS expression). Eng et al (55) reported that both NIS mRNA and NIS protein levels decreased sig nificantly after either 1 or 6 days of I- administration in rats. NIS mRNA levels were already significantly reduced at 6 hours after injection of a single dose of I-. In contrast, a significant decrease of NIS protein levels was detected at only 24 hours. These findings were not correlated with NIS activity by thyroid scintigraphy. The conclusion of this study was that the decrease in active I- transport, that is, the basis for the “escape,” occurs between 6 and 24 hours by a mechanism that at least in part involves a decrease in NIS transcription.
Eng et al (56) later investigated the effect of I- on NIS mRNA and protein expression in FRTL-5 cells. Incubation of FRTL-5 cells with I- (10-3 M) did not affect NIS mRNA levels, but NIS protein levels decreased significantly in a dose-dependent manner. This conflicts with the authors' previous in vivo observations (55) and with the findings of Spitzweg et al (57), who reported a 50% decrease in NIS mRNA levels in FRTL-5 cells incubated with I- (10-4 M). When I- was administered during TSH stimulation (72 hours after TSH deprivation), the increase in NIS protein levels was less pronounced in the I- -treated cells than in the controls. Performing pulse-chase experiments, the authors found that the half-life of the NIS protein was shorter in the I- -treated cells, suggesting increased NIS protein turnover in these cells. However, the half-life of NIS indicated in this study in normal untreated FRTL-5 cells was < 24 hours, which is much shorter than the 4 to 5 days reported by several other groups (18,20,34,47). In summary, the authors (50,56) concluded that high doses of I-administered in vivo lead to decreases in both NIS mRNA and protein levels by a mechanism that is likely to be at least in part transcriptional, whereas their studies in vitro suggested that the I- -induced decrease in NIS protein levels appears to be due at least in part to an increase in NIS protein turnover.
Clearly, NIS regulation is complex. Whereas NIS is located both at the plasma membrane and in intracellular organelles, I- uptake is mediated only by NIS molecules at the plasma membrane (32,47). The subcellular distribution of NIS is regulated mainly by TSH (32,47). Hence, studies parallel of NIS mRNA and protein expression, and the cellular distribution and function of NIS, both in vitro and in vivo, are needed to better understand the regulatory effects exerted by I- on NIS.
Sodium-Iodide Symporter Regulation by Thyroglobulin
As discussed earlier, NIS activity is up-regulated by TSH. Kohn's group (58) reported the intriguing observation that Tg acts as a potent suppressor of NIS mRNA levels and thyroid-restricted genes (i.e., Tg, TPO, and TSHR) in FRTL-5 cells, and suggested that Tg could counterbalance the effect of TSH on these genes. The notion of Tg acting as a NIS suppressor is surprising because of the characteristics of the Tg molecule. Tg is synthesized as a 12S molecule that forms a 19S dimer and a 27S tetramer (59,60). Using 19S follicular Tg (at concentrations known to exist in the follicular lumen) these researchers (61) indicated that follicular Tg suppressed TSH-increased NIS activity in vitro and in vivo, and regulated the Tg, TPO, and TSHr genes at the transcriptional level (61). Purified 12S, 19S, and 27S follicular Tg suppression of thyroid-restricted gene expression was dependent on the ability of the Tg to bind to thyrocytes (62). This binding was blocked by an antibody against the thyroid apical membrane asialoglycoprotein receptor, a phosphoprotein that is critical for ATP-mediated inactivation of receptor-mediated endo cy tosis (62).
CURRENT AND FUTURE CLINICAL RELEVANCE OF THE SODIUM-IODIDE SYMPORTER
Sodium-Iodide Symporter in Thyroid Cancer
Thyroid cancer remnants and metastases have long been successfully treated with radioiodide, after thyroidectomy. Although I- uptake in most thyroid cancers is decreased relative to the surrounding normal tissue, uptake is still sufficient to enable administered radioiodide to destroy remnant tumor cells and metastases. A clear understanding of the cause of diminished I- uptake in thyroid cancer will certainly have major implications for the treatment of this disease. The fundamental issue is whether I- uptake is reduced in thyroid cancer cells as a result of a decrease in NIS expression, or by some other mechanism, such as a defect in NIS targeting to the plasma membrane. NIS expression in thyroid cancer has been investigated at both the RNA and protein levels by several groups (63,64,65,66,67,68). First, no mutations were found in NIS by direct sequencing by PCR of NIS cDNA obtained from papillary carcinomas, ruling out that a mutation might be impairing NIS function in these tumors (66). Second, variable levels of NIS mRNA expression in thyroid carcinomas have been found by molecular biologic methods, which (63,64,65,66,67,68) provide information only on the level but not the stability of the NIS mRNA. Given that there is great interest in increasing NIS function in thyroid cancer, some investigators have attempted to induce NIS expression in thyroid carcinoma cell lines by means of demethylation treatment and with transretinoic acid. However, the results have been inconclusive (69,70).
Since NIS is a membrane protein with a long half-life (19) and with a complex transcriptional, posttranscriptional, and posttranslational regulation, the presence or absence of the NIS transcript and the levels observed are not sufficiently revealing on the levels of expression of the NIS protein, and even less on whether NIS is properly targeted to the plasma membrane, the only location where it is functional. It is relevant to point out that the coexistence of marked differences in mRNA and protein levels has been reported with such other proteins as the transferrin receptor (71,72). The expression of the NIS protein can be ascertained by immunoblot analysis and immunohistochemistry. Whereas immunoblot analysis does provide information on the expression of the NIS protein, it is a technique that requires a relatively large amount of tissue, which is rarely available, and has the disadvantage that it does not reveal the subcellular localization of the protein. Immunohistochemistry, does give an indication of the subcellular localization of NIS. Saito et al (73) investigated papillary and follicular cancers by immunoblot analysis and immunohistochemistry, and reported overexpression of the NIS protein in the majority of tumors. Dohán et al (74) have also investigated NIS protein expression by immunohistochemistry in a large sample of differentiated thyroid carcinomas and found that as many as 70% overexpressed NIS protein compared to the surrounding normal tissue (Fig. 4A.6). Significantly, NIS was localized mainly intracellularly in most of the tumors, strongly suggesting that the decrease in I- uptake in most thyroid carcinomas is not due to low NIS expression but to alterations in NIS trafficking (Fig. 4A.6) (74). This was somewhat surprising, because a lower NIS expression could have been expected in the face of the established lower NIS activity in these cells.
FIGURE 4A.6. Immunohistochemical analysis of sodium-iodide symporter (NIS) protein expression in thyroid carcinomas. A: Normal thyroid (400×magnification). B: Thyroid tissue from a patient with Graves' hypothyroidism showing a distinct basolateral staining pattern (1,000× magnification). C: Papillary carcinoma tissue showing strong intracellular staining (1,000× magnification). D: Papillary carcinoma with negative NIS expression (400× magnification).
The finding that the NIS protein is actually overexpressed in thyroid cancer cells suggests that the mechanism for the decrease in activity is not a simple impairment of NIS protein expression. These results have been confirmed and extended by Wapnir et al (75), who analyzed a very large number of tissue microarrays and conventional sections and found NIS protein expression in 75% of benign thyroid nodules and 73% of thyroid cancers. Furthermore, Tonacchera et al (76) have reported that 54% of benign nonfunctional thyroid nodules overexpressed hNIS protein, as compared to normal surrounding tissue; significantly, NIS was located intracellularly in these nodules as well.
In short, currently available data indicating that the decrease of I- transport activity coexists with NIS overexpression, but with a predominantly intracellular localization, underscore the importance of elucidating the molecular mechanisms underlying the proper targeting to and retention of NIS at the plasma membrane.
To be therapeutically effective, radioiodide has to remain within the thyroid cancer cells for a sufficiently long time. The major determinants of I- retention in thyroid cells are NIS-mediated I- uptake, I- organification, and I- efflux. Based on the thus far sparse investigations on I- organification in thyroid cancer, it seems that organifi cation is impaired in these tumors. Several groups have tried to identify the mediator of apical I- efflux in thyroid epithelial cells. Two recently cloned molecules are the main candidates, pendrin and apical iodide transporter (AIT), although neither has been conclusively proven to be the mediator of apical I- efflux.
In 1997, a gene defective in Pendred's syndrome (PDS) was identified by positional cloning (77). PDS is charac terized by sensorineural (most often prelingual) deafness and goiter with defective I- organification. In PDS, goiter can develop at any age or may be absent, whereas deafness is generally present (78). Pendrin has been localized on the apical membrane of the thyroid epithelial cells by immunohistochemistry (79). In heterologous expression systems, pendrin has been shown to transport iodide, chloride, formate, and nitrate (14).
The organification defect characteristic of PDS was attributed to defective pendrin-mediated apical iodide transport into the colloid, where organification occurs (Fig. 4A.1). Surprisingly, although Pds-knockout mice are completely deaf, they do not have a pathologic thyroidal phenotype (78); therefore, pendrin's function as the AIT remains to be further investigated.
Using a cloning strategy based on NIS sequence ho mologies, a 610 amino acid protein-coding gene was recently cloned from a human kidney cDNA library. The newly identified protein shares both a strikingly high identity (46%) and similarity (70%) to hNIS (15). This protein, called the human AIT (hAIT), has been localized to the apical membrane of thyroid epithelial cells; however, a thorough molecular and kinetic characterization is required to unequivocally establish whether hAIT mediates “downhill” movements of I- from the cytosol to the colloid.
Radioiodide is the cornerstone of the treatment of metastatic thyroid cancer. The optimal therapeutic radioiodide dose is calculated on the basis of the scintigraphic image obtained upon administration of a radioiodide test dose. This test dose must be properly adjusted so as to prevent uptake inhibition of the subsequently administered therapeutic dose of 131I-. The interference of radioiodide test doses with uptake of subsequent therapeutic doses is called “stunning,” the molecular mechanism of which is unknown (80) (see Chapters 12).
To investigate stunning, Nilsson et al (81) exposed pig thyrocyte primary cultures [grown in a bicameral chamber, where vectorial (basal to apical) I- transport can be assessed] to increasing doses of 131I- or stable, nonradioactive 127I- (1 to 100 Gy, iodide < 10-9 M) for 48 hours in the presence of TSH and methimazole (MMI). Basal to apical I- transport was then measured using 125I-. Immediately after exposure to radioiodide, active I- transport was similar to the control. However, 3 days after 131I- exposure, basal to apical I- transport decreased in a radioiodide dose-dependent manner. Based on these observations, the authors concluded that stunning of I- accumulation after radio iodide exposure is due to selective inhibition of the I- transporting mechanism.
Iodide Transport Defect
The molecular analysis of some of the NIS mutations detected in patients with congenital I- transport defect has provided important structural information about the symporter. Iodide transport defects—caused by NIS mutations—are conditions characterized (if untreated) by hypo thyroidism, goiter, low thyroid I-uptake, and low saliva/ plasma I- ratio. Eleven NIS mutations have been identified so far: V59E, G93R, Q267E, C272X, G395R, T354P, frame-shift 515X, Y531X, G543E, d 143 to 323, and δ 439 to 443 (82,83,84,85,86,87,88,89,90,91). They are either nonsense, alternative splicing, frame-shift, deletion, or missense mutations of the NIS gene (Fig. 4A.2).
The T354P, G395R, and Q267E NIS mutant molecules have been investigated at the molecular level. Both mutant T354P and G395R NIS proteins are synthesized and properly targeted to the plasma membrane. The detailed molecular analysis of mutation T354P, carried out by Levy et al (92), revealed that a hydroxyl group at the β-carbon at position 354 is essential for NIS function. Dohán et al (93), thorough structure/function analysis of the G395R mutation, showed that the presence of an uncharged amino acid residue with a small side chain at position 395 is required for NIS function, and that the presence of charge or of a long side-chain at position 395 results in decreased turnover rate of the transporter, without affecting its ion-binding affinity. Based on flow cytometry data, it was at first suggested that the Q267E mutation results in impaired plasma membrane trafficking of the mutated NIS protein (94). However, De la Vieja et al (95), using several experimental approaches, have recently shown that the Q267E mutant NIS is modestly active and properly targeted to the plasma membrane. The mutant protein has a lower Vmax for iodide as compared to the wild-type protein, which could result in the lower transport rate. In contrast to T354P, G395R, and Q267E, G543E NIS matures only partially, and it is the only mutant identified thus far that is not targeted properly to the cell surface, apparently because of faulty folding (95). Therefore, the G543 resi due plays significant roles in NIS maturation and trafficking. Remarkably, NIS activity was rescued by small neutral amino acid substitutions at this position, suggesting that G543 is in a tightly packed region of NIS. Clearly, the detailed molecular analysis of NIS mutations will con tinue to reveal substantial structure/function information on NIS.
NIS in Extrathyroidal Tissues
The field of I- transport systems outside the thyroid has changed considerably since the extensive review published on the topic in 1961 by Brown-Grant (96). The main vertebrate nonthyroid tissues reported to actively accumulate I- are salivary glands, gastric mucosa, and lactating mammary gland (Fig. 4A.7). Many of these transport systems exhibit functional similarities with their thyroid counterpart, notably a susceptibil ity to in hibition by thiocyanate and perchlorate. How ever, they also display important differences: (a) non thyroid I- transporting tissues do not have the ability to or ganify accumulated I- (with the possible excep tion of the lactating mammary gland); therefore, they behave like MMI-treated thyroid tissue; (b) TSH exerts no regulatory influence on nonthyroid I-accumulation; (c) at least salivary glands and gastric mucosa concen trate thiocyanate, unlike the thyroid, where thiocyanate is metabolized after uptake and therefore not concentrated.
FIGURE 4A.7. Immunohistochemical analysis of sodium-iodide symporter protein expression in tissues that exhibit active I- transport. Shown from top to bottom are thyroid, salivary gland, stomach, and lactating mammary gland.
NIS is clearly regulated and processed differently in each tissue. The cloning of human NIS cDNAs has been reported from gastric mucosa, parotid glands, and mammary glands, all of which exhibited full identity to thyroid hNIS cDNA. Whereas hNIS gene expression has been detected in many other tissues by (27,97,98,99,100,101,102), the molecular biologic techniques used yield a large number of false positives due to their high sensitivity (103). Therefore, the detection of the NIS amplified product in a given tissue cannot be regarded as sufficient evidence that NIS is functionally expressed in that tissue. A thorough characterization of NIS protein expression is necessary to properly evaluate the significance of results obtained by these techniques. Still, even with the use of a wide variety of techniques (Northern analysis, DWA amplification, Western analysis, and immunohistochemistry), different groups have often obtained inconsistent and sometimes conflicting results on whether or not NIS is expressed in a particular tissue. Hence, once NIS protein expression has been demonstrated, a correlation with Na+-dependent, perchlorate-sensitive, active I- accumulation in that tissue must be established. By these criteria, and taking into consideration the results mentioned earlier, NIS is expressed and active in extrathyroidal tissues previously known to exhibit NIS activity, such as salivary glands, gastric mucosa, and lactating mammary gland. NIS expression in different tissues is shown in Fig. 4A.8. Electrophoretically, NIS migrates as a broad band, which is characteristic of extremely hydrophobic, polytopic membrane proteins. The significance of the detection of the amplified NIS product in other human and rat tissues remains to be ascertained.
FIGURE 4A.8. Immunoblot analysis of sodium-iodide symporter (NIS)-expressing cell lines (lanes 1 through 5) and human tissues (lanes 6 through 9). Lane 1: FRTL-5 cells. Lane 2: FRT cells. Lane 3: COS-7 cells transiently transfected with rat NIS (rNIS) cyclic DNA (cDNA). Lane 4: COS-7 cells transiently transfected with human NIS cDNA. Lane 5: MCF-7 cells. Lane 6: Thyroid. Lane 7: Salivary gland (parotid). Lane 8: Breast tissue from a pregnant woman in the third trimester. Lane 9: Gastric mucosa. Membrane fractions from the cell lines or from homogenized tissues were separated by 9% SDS-PAGE, then electroblotted to nitrocellulose, and probed by 0.5 µg/mL affinity-purified polyclonal anti-rNIS antibodies (Ab) (lanes 1 through 3) or by hNIS Ab (lanes 4 through 9), followed by horseradish-peroxidase (HRP)-labeled goat anti-rabbit Ab. Immunoreactive bands were visualized by enhanced chemiluminescence.
Sodium-Iodide Symporter in Breast Cancer
As indicated earlier, NIS is differently regulated and subjected to distinct posttranslational modifications in each tissue where it is expressed (104,105,106). Physiologically, I- transport in the mammary gland occurs during late pregnancy and lactation, resulting in the transfer of I- to the milk. An adequate supply of I- for sufficient thyroid hormone production is essential for proper development of the newborn's nervous system, skeletal muscle, and lungs. In contrast, no I- transport is observed in normal breast tissue in the absence of pregnancy and lactation.
Using in vivo scintigraphic imaging and immunoblot analysis, Tazebay et al (106) demonstrated functional expression of NIS in experimental mammary adenocarcinomas in nongestational and nonlactating female transgenic mice carrying either an activated ras oncogene (c-Ha-ras) or overexpressing the Neu oncogene (c-erbB-2) (107). When Tazebay et al (106) studied through immunohistochemical analysis human breast tissue specimens for NIS expression, they found that 87% of the invasive breast cancers and 83% of the ductal carcinomas in situ expressed NIS, as compared with only 23% of the extratumoral samples adjacent to or in the vicinity of the tumors and to none of the normal samples from reductive mammoplasties (76). More recently, Wapnir et al (75) examined the immunohistochemical profile of NIS in thyroid, breast, and other carcinomas using high density tissue microarrays and conventional sections. The results confirmed the findings stated above: NIS expression was demonstrated in whole tissue sections in 76% of invasive breast carcinoma and 88% of ductal carcinoma in situ samples. The major ity of normal breast cores were negative (87%), as were 70% of normal/nonproliferative samples analyzed. Plasma membrane immunoreactivity was observed in gestational breast tissues, and in some in situ ductal carcinomas and invasive ductal carcinomas.
Because all the above studies in human samples were carried out by immunohistochemistry, the findings demonstrate only NIS expression, but not necessarily NIS functional expression in human breast cancer. Hence, in a significant first step addressing this issue, Moon et al (108) have reported pertechnetate accumulation in primary breast tumors in humans in vivo. Pertechnetate (99mTcO4) is a radioisotope widely used for diagnostic imaging, also transported by NIS, with the advantage of having a shorter half-life (t1/2 6 hours) than 131I (t1/2 8 days). These authors studied 25 cancer patients by scintigraphy and found active uptake by the tumors in 4 patients. This is a highly meaningful result, not only because it demonstrates the existence of I- transport activity in a significant percentage of human breast cancer patients in vivo, but also because the observation was made in patients whose thyroid glands were not down-regulated (i.e., in these patients thyroid NIS was still expressed, and therefore there was avid thyroidal I- uptake; this decreased the amount of radioisotope available for uptake by breast tumors). It is possible that a larger proportion of I--accumulating breast cancer tumors might have been detected by scintigraphy if the availability of the radio isotope to tumoral tissue had been optimized by thyroid suppression.
In conclusion, although more extensive studies in humans are necessary, functional expression of NIS in breast cancer has been documented both in mice and humans. This suggests strongly that NIS is up-regulated with high frequency during malignant transformation in breast cancer. Therefore, endogenous NIS has tremendous potential to serve as the conduit for radioiodide in the diagnosis and treatment of this devastating disease.
Sodium-Iodide Symporter Gene Transfer
Given the high efficacy and low rate of side effects of NIS-mediated radioiodide therapy in thyroid cancer, it is extremely desirable to develop strategies to apply this therapy in other cancers. For this purpose, cancers may be divided into two groups, namely those that express endogenous NIS (with full, partial, or no activity) and those that do not. To date, the only cancer other than thyroid cancer shown to express functional endogenous NIS molecules is breast cancer. For both thyroid and breast cancer, it appears to be advantageous to identify ways to increase activation of endogenous NIS, such as by prodding more NIS molecules to be targeted to the plasma membrane instead of being retained in intracellular compartments (47). For cancers that do not express endogenous NIS, ectopic NIS may be introduced by a variety of gene transfer techniques, as explained later in the chapter (109,110,111).
Whether a tumor expresses functional NIS endogenously or ectopically (by gene transfer), NIS function makes it possible to image, monitor, and treat the tumor with radioiodide, just as in differentiated thyroid cancer. Compared with other genes that have been transferred, NIS offers the unique advantage that it can be used both as a reporter and a therapeutic gene, as has been shown by in vivo imaging of NIS-bearing tumors in various animal models (109,110,111,112). Functional expression of NIS can be detected by using available techniques to monitor the radioac tive tracers transported by NIS, such as 125I, 123I, 124I, or 99mTcO-4). Each of these tracers offers its own advantages and disadvantages in terms of its half-life, availability, price, and so on.
Human and rat NIS have been introduced into several different tumor cell lines under the control of tissue-specific or general promoters using different transfer meth odologies, such as liposomal transfection, electroporation, or adenoviral or retroviral transduction (104,105). As ex pected, functional NIS expression was detected in vitro in these cells. All authors concluded that in vivo gene therapy experiments have to be the next step. However, the most difficult problem to overcome is the targeted deliv ery of the NIS gene to the tumors in vivo. Thus far, three in vivo studies in mice have yielded promising results (109,110,111).
Using NIS-containing recombinant adenovirus, Cho et al (109) have recently shown that hNIS can be functionally expressed in xenografted human glioma in rats. These authors observed significant radioiodide retention in the tumors over 24 hours. They administered 3 × 4 mCi (in 2-day intervals) of 131I for 2 weeks following tumor implantation, and observed longer survival in animals bearing NIS-expressing tumors. Spitzweg et al (114) introduced NIS in a recombinant adenovirus into a human prostate carcinoma cell line under the regulation of the prostate specific antigen promoter. They reported a mean retention time of 5.6 hours in NIS-expressing prostate carcinoma xenografts in nude mice and a remarkable decrease (>80%) of the size of these xenografts after a single intra peri toneal injection of 3 mCi 131I. Dingli and colleagues (110) expressed NIS in a myeloma cell line using transcrip tionally targeted lentiviral vector, where the therapeutic or reporter gene is under the control of minimal immunoglobulin promoter and enhancer elements (immunoglobulin k-light chain enhancer elements). These authors also investigated the so-called bystander effect. β-particles emitted during the decay of 131I can travel a distance of 0.2 to 2.4 mm. Therefore, the isotope is capable of destroying the “bystanding” non-NIS-expressing cells. Dingli et al (110) also treated myeloma xenografts containing variable numbers of radioiodide-transporting, NIS-transduced and nontransduced tumor cells. The result was striking: all tumors in which 50% to 100% of the cells expressed NIS completely regressed 2 weeks after a single dose of 1 mCi 131I.
The above results provide strong evidence against the widely held notion that radioiodide therapy is likely to be ineffective in nonthyroidal cells that, while functionally expressing NIS (whether endogenously or by targeted transfection), lack the ability to organify I-. The reasoning was that the absence of organification resulted in the isotope not being retained in the cells for a sufficiently long time. Yet, in several studies (109,110,111,112,113), radioiodide treatment was effective even in the absence of I- organification.
The therapeutic potential of other radioisotopes transported by NIS is currently under investigation. Astatide (211At), an α-emitter with high linear energy transfer (97 keV/µ M) and a short tissue range (79 µm), has been shown to be transported by NIS in a perchlorate-sensitive manner in the UVW human glioma cell line stably transfected with hNIS cDNA (114).
188ReO4, a β-emitter (Eaverage = 0.764 MeV) (131I E = 0.134 MeV, optimal tissue range: 2.6 to 5 mm), has been suggested as an alternative to 131I for breast cancer treatment. ReO4 was not transported in NIS-expressing X. laevis oocytes (22); however, Van Sande et al (115) have reported its accumulation in FRTL-5 cells and in COS-7 cells stably transfected with hNIS. In MDCK cells permanently transfected with hNIS, it has been shown that the Km for ReO4 is similar to that of iodide, but the Vmax and consequently the accumulated isotope in the cells is about half (116). These new NIS-transported radioisotopes can be potentially useful in various clinical settings, but for now we lack the experience in using them the same way we do after having accumulated over 60 years of employing radioiodide in thyroid cancer.
CONCLUDING REMARKS AND FUTURE PERSPECTIVES
The thyroid's ability to actively take up I-, first detected as early as the 19th century, has long been regarded as one of the most distinctive and significant attributes of the gland, even before NIS was identified as the thyroid I- transporter. For over six decades, this ability has been key to the evaluation of thyroid function and the diagnosis and treatment of thyroid disease. Once it was identified, NIS was quite naturally expected to prove to be a thyroid-specific protein with major physiological and medical significance, probably not expressed in any other tissues. As NIS was demonstrated to be the single mediator of active I-uptake in several other I- -transporting tissues, most notably the lactating mammary gland, it becomes apparent that NIS may have medical applications beyond the thyroid. Remarkably, NIS is now seen as the key to extend the use of radioiodide and possibly related isotopes to a wide variety of cancers beyond the thyroid, as effective anticancer therapy. The discovery that endogenous NIS is functionally expressed in breast cancer, especially, places NIS squarely at the center of some of the current efforts to combat the deadliest malignancy in women. In addition, available findings on the transfer of the NIS gene to a variety of cancers, from prostate to glioma, suggest that radioiodide may also be eventually used against cancers that do not express NIS endogenously. There is no question that the continued elucidation of NIS structure/ function relations, regulation, and mechanistic information will provide ad ditional insights of ample physiological and pathophysiological importance.
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