help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1562
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
90/4/2321    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arturi, F.
Right arrow Articles by Filetti, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arturi, F.
Right arrow Articles by Filetti, S.
Related Collections
Right arrow Endocrine Oncology
Right arrow Thyroid
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 4 2321-2326
Copyright © 2005 by The Endocrine Society

Regulation of Iodide Uptake and Sodium/Iodide Symporter Expression in the MCF-7 Human Breast Cancer Cell Line

Franco Arturi1, Elisabetta Ferretti1, Ivan Presta, Tiziana Mattei, Angela Scipioni, Daniela Scarpelli, Rocco Bruno, Ludovic Lacroix, Emanuele Tosi, Alberto Gulino, Diego Russo and Sebastiano Filetti

Dipartimento di Medicina Sperimentale e Clinica (F.A., I.P., D.S.) and Dipartimento di Scienze Farmacobiologiche (D.R.), Università di Catanzaro, "Magna Graecia," 88100 Catanzaro, Italy; Dipartimento di Medicina Sperimentale e Patologia (E.F., A.G.) and Dipartimento di Scienze Cliniche (T.M., A.S., E.T., S.F.), Università "La Sapienza," 00161 Roma, Italy; Tinchi Pisticci Hospital (R.B.), 75020 Matera, Italy; and Department of Clinical Biology (L.L.), Institut Gustave-Roussy, 94805 Villejuif Cedex, France

Address all correspondence and requests for reprints to: Sebastiano Filetti, M.D., Università degli Studi di Roma La Sapienza, Dipartimento di Scienze Cliniche, Clinica Medica 2, Viale del Policlinico, 155, 00161 Roma, Italy. E-mail: sebastiano.filetti{at}uniroma1.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sodium/iodide symporter (NIS) expression has recently been described in human breast cancer, with emphasis on its potential exploitation for the treatment of these tumors with radioiodine. In this study, we analyzed the regulation of NIS expression and function in the MCF-7 human breast cancer cell line. Cell exposure to insulin, IGF-I, IGF-II, or prolactin induced significant increases in 125I uptake and the expression of both NIS mRNA and NIS protein. The latter increases were evident after 6 and 12 h of hormonal stimulation, respectively. In immunocytochemistry studies, NIS was detected mainly in the plasma membrane of MCF-7 cells. A low but significant increase in iodide uptake was produced by treatment with activators of the adenylyl cyclase (cAMP) or protein kinase C pathways. Our study demonstrates that: 1) MCF-7 breast cancer cells are capable of active iodide transport that can be stimulated by insulin, IGF-I, IGF-II, or prolactin; 2) both NIS transcript and protein are expressed in these cells, and this expression is also hormonally stimulated; and 3) MCF-7 iodide transport and NIS expression may be influenced by the activation of cAMP or protein kinase C-dependent signaling. These findings increase our understanding of the molecular mechanisms that regulate NIS expression in breast cancer cells, information that is fundamental for future research aimed at the development of targeted radioiodide treatment for this type of cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ABILITY TO concentrate iodide is a property of normal thyroid tissue, and its persistence in thyroid cancer cells is a fundamental prerequisite for the use of radioiodine in the diagnosis and treatment of patients with differentiated thyroid carcinomas (1). Characterization of the sodium/iodide symporter (NIS), the protein responsible for iodide transport across the basal membrane of the thyrocyte (2, 3), has provided new insights into the molecular mechanisms underlying the loss or reduction of iodide trapping in certain thyroid cancer cells (4, 5, 6, 7, 8, 9, 10).

Several studies have demonstrated that NIS expression and radioiodine uptake activity also occur in various extrathyroidal tissues (9, 10, 11, 12, 13), including the lactating mammary gland (14, 15, 16, 17). In the latter tissue, physiological iodide transport occurs exclusively in the later stages of pregnancy and during lactation. Its purpose is to provide the nursing newborn with an essential source of iodide for the biosynthesis of thyroid hormones (12). Tazebay et al. (17) found the NIS protein in the mammary tissues of pregnant mice shortly before they gave birth and later while they were nursing. Its expression decreased significantly when the pups were being weaned, but high levels returned if suckling resumed. This observation suggests that iodide trapping in breast cells is mediated mainly by the NIS and the expression of this transporter is hormonally regulated, in large part by the hormones present during lactation [oxytocin and prolactin (PRL)] (17).

NIS mRNA and protein have also been identified in the majority of human breast cancer cells (16, 17, 18). These findings raise the intriguing possibility that radioiodide might someday be used for the detection of breast malignancies and their targeted destruction (17, 19, 20). Before this possibility can become a reality, however, a number of fundamental issues must be addressed. One of these regards the molecular mechanisms involved in the regulation of NIS expression and function in breast cancer cells. In the present study, we investigated the hormonal regulation of iodide transport and NIS expression in a well-established model of human breast cancer, the MCF-7 cell line.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures and test substances

Cells of the MCF-7 human breast cancer line (obtained from the American Type Culture Collection, Manassas, VA) were grown in DMEM (Sigma-Aldrich S.r.l., Milan, Italy), supplemented with 10% fetal bovine serum (FBS) (Life Technologies S.r.l., San Giuliano Milanese, Milan, Italy) and containing penicillin/streptomycin and amphotericin B (Sigma-Aldrich S.r.l.). Each experiment was initially conducted on cells that had been maintained for 5 d in culture medium, supplemented only with 0.2% FBS (basal conditions). Assays were then repeated after the cells had been exposed for 2, 6, 12, and 24 h to insulin, IGF-I, and IGF-II (10–8 M) (Sigma-Aldrich, St. Louis, MO), or PRL (500 ng/ml PRL) (R&D System Europe Import-Export S.r.l., Milan, Italy). In other experiments, cells were treated with 17-ß-estradiol, oxytocin, KClO4, forskolin, dibutyryl cAMP [(Bu)2-cAMP], ouabain, 12-O-tetradecanoyl phorbol 13-acetate (TPA), or cycloheximide (all purchased from Sigma-Aldrich). FRTL-5 cells and CHO cells, grown as described previously (21), were used as positive and negative controls, respectively.

Measurement of 125I uptake

Uptake of 125I by MCF-7 cells was measured as described previously (21, 22). Briefly, cells were split and seeded into 12-well plates. The culture medium (described above) was aspirated, and the cells were washed with 1 ml of Hank’s balanced salt solution (HBSS) (Life Technologies S.r.l.) supplemented with HEPES 10 mM (pH 7.3). 125I uptake was initiated by adding to each well 500 µl of buffered HBSS, containing 0.1 µCi carrier-free Na125I and 10 µM NaI (specific activity, 20 mCi/mmol). In half of the wells, this assay buffer also contained the NIS inhibitor, KClO4 (100 µM), as a control for nonspecific uptake. After 40 min, at 37 C in a humid atmosphere, the radioactive medium was aspirated, and cells were washed with 1 ml of ice-cold HBSS. One milliliter of 95% ethanol was placed in each well for 20 min, and then transferred into vials for gamma counting to measure the amount of 125I associated with the cells. Iodide uptake was expressed as picomoles per microgram of cellular DNA, which was measured with a fluorometric DNA assay kit (Bio-Rad Laboratories, Segrate, Milano, Italy). Each experiment was performed at least twice in quadruplicate, and FRTL-5 and CHO cells were included as positive and negative controls, respectively.

Determination of mRNA level using real-time RT-PCR

Total RNA was extracted from the cells using the RNA Fast Kit (M-Medical S.p.A, Florence, Italy) according to the manufacturer’s instructions. Two micrograms of RNA were then reverse transcribed in a 20-µl reaction volume containing 200 U of Moloney murine leukemia virus reverse transcriptase; 40 U of ribonuclease inhibitor; 10 mmol/liter of dA/T/C/G; 3 mM of MgCl2; 50 mM of Tris-HCl (pH 8.3); 75 mM of KCl; and 600 ng of random hexamers (Invitrogen, Paisley, UK). The cDNAs were then diluted 1:10 in nuclease-free H2O (Life Technologies, Inc., Milan, Italy) and analyzed by real-time PCR in an ABI Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA), which scans 96 sample tubes per assay. In accordance with the manufacturer’s instructions, each tube (reaction mixture volume, 25 µl) contained 2.5 µl of cDNA, 12.5 µl TaqMan Universal PCR master mix (Applied Biosystems), 200 µM TaqMan probe, and 900 µM of each primer for the NIS gene. The following thermal cycler parameters were used: incubation at 50 C for 2 min and denaturing at 95 C for 10 min, then 40 cycles of amplification (each consisting in denaturation at 95 C for 15 sec and annealing/extension at 60 C for 1 min). A standard curve was generated for each amplification, using six serial dilutions of a cDNA mix expressing NIS. All amplification reactions were performed in triplicate, and the average of the three threshold-cycle values was used. Sequence Detection System Software (version 1.7, Applied Biosystems) was used for construction of standard curves and relative quantification of transcript in samples. mRNA quantification results were expressed as the ratio of the target quantity to the quantity of the calibrator, a sample of unstimulated MCF-7 cells. All values were normalized to two endogenous controls, glyceraldehyde-3-phosphate dehydrogenase and ß-actin, with similar results. Primers and probe sets used for control genes were TaqMan predesigned assay reagents (Applied Biosystems).

Protein extraction and Western blot analysis

Total proteins were extracted from MCF-7 cells as described previously (7). Briefly, confluent cells from three different plates (90 mm in diameter) were homogenized in 1 ml of buffer containing 250 mM sucrose, 10 mM HEPES-KOH (pH 7.5), 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The homogenate was centrifuged at 14,000 x g (4 C for 15 min), and the supernatant (which contained the whole-cell lysate) was analyzed spectrophotometrically, using the Bradford method (23). Thirty micrograms of proteins were then loaded onto an 8% polyacrylamide gel and subjected to electrophoresis at a constant voltage (110 V). Electroblotting to Hybond-P ECL nitrocellulose membranes was performed overnight at 15 V, using a Mini Trans-Blot system. Blocking was carried out for 1 h at room temperature (RT), using Dulbecco’s PBS/5% nonfat dry milk (PBS/milk). The membrane was then incubated for 90 min in PBS/milk, with a 1:1000 dilution of affinity-purified rabbit anti-NIS polyclonal antibody (24), or with a 1:3000 dilution of mouse monoclonal antihuman ß-actin antibody (Sigma-Aldrich S.r.l.). After three 5-min washes in PBS, the membrane was incubated with a 1:2000 dilution of horseradish peroxidase-conjugated antirabbit or antimouse antibody (DBA Italia S.r.l., Segrate, Milan, Italy) in PBS/milk. After three 5-min washes in PBS, the protein was visualized with an enhanced chemiluminescence Western blot detection system (ECL from Amersham Pharmacia Biotech) and quantified densitometrically.

Immunocytochemistry study

Cells were fixed in 4% paraformaldehyde for 10 min at RT. After three 5-min washes in PBS, the cells were incubated in blocking buffer (1x PBS, 1% BSA, and 0.05% Triton X-100) for 15 min at RT. After three 5-min washes in PBS, the cells were incubated with a 1:1500 dilution of affinity-purified rabbit anti-NIS polyclonal antibody (25) for 1 h at RT and rewashed in PBS. They were then incubated with a 1:400 dilution of fluorescein isothiocyanate antirabbit antibody (Sigma Aldrich S.r.l.) for 30 min at RT, rewashed in PBS, and incubated in Hoechst 1x for 3 min at RT. After three final PBS washes (5 min each), the cells were mounted with Vectaschield (Vector Laboratories, Inc., Burlingame, CA).

Statistical analysis

Results are expressed as mean ± SD. Differences between results observed at different times during stimulation were analyzed by one-factor ANOVA, followed by t test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of insulin, IGF-I, and IGF-II on 125I uptake by MCF-7 cells

Low-level 125I uptake (1.50 ± 0.5 pmol/µg DNA) was detected in MCF-7 cells maintained for 5 d in growth medium supplemented with 0.2% FBS (basal conditions). This activity was specific and was inhibited by the NIS blocker, perchlorate (0.4 ± 0.1 pmol/µg DNA). Six hours after the addition of insulin, IGF-I, or IGF-II (10–8 M), 125I uptake increased significantly and reached maximum levels after 12 h of stimulation (12-fold, 7.8-fold, and 10.3-fold increases over basal levels, respectively) (P < 0.001) (Fig. 1Go, A–C). The amount of iodide incorporated by MCF-7 cells stimulated with insulin, IGF-I, or IGF-II was roughly half that observed in control FRTL-5 cells stimulated with TSH (15.2 ± 2.0 pmol/µg DNA vs. 32.0 ± 3.6 pmol/µg DNA). Stimulated (steady-state) uptake was inhibited by perchlorate (Table 1Go) and by the Na+-K+-ATPase inhibitor, ouabain (data not shown).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 1. Effects of insulin, IGF-I, IGF-II, and PRL on iodide uptake in MCF-7 cells. Iodide uptake was measured, as described in Materials and Methods, under basal conditions and at different times after the addition (time 0) to growth medium of insulin, IGF-I, IGF-II (each used at a concentration of 10–8 M), or PRL (500 ng/ml). Iodide uptake is expressed as the mean ± SD of values obtained in three different experiments (n = 3). *, P < 0.05; **, P < 0.001 (ANOVA followed by t test).

 

View this table:
[in this window]
[in a new window]
  		TABLE 1. Effects of perchlorate (KClO4) and cycloheximide (CH) on iodide uptake induced in MCF-7 cells by stimulation with insulin, IGF-I, IGF-II, or PRL

 
Experiments were then repeated with MCF-7 cells grown in medium containing 10 µg/ml of cycloheximide, an inhibitor of protein synthesis. Under these conditions, the stimulation of iodide uptake induced by insulin, IGF-I, or IGF-II was diminished but not completely abolished, suggesting that the effects of these hormones are partially mediated by new protein synthesis.

Effects of PRL, forskolin, (Bu)2-cAMP, TPA, 17-ß-estradiol, and oxytocin on 125I uptake by MCF-7 cells

PRL produced dose-dependent increases in iodide accumulation in MCF-7 cells (Fig. 2Go). As shown in Fig. 1DGo, 125I uptake was significantly increased after 6 h of stimulation with PRL (500 ng/ml), and the maximum effect (9-fold increase over basal levels) (P < 0.001) was observed after 12 h. The PRL-stimulated uptake, like that produced by insulin and IGFs I and II, was perchlorate-sensitive and significantly decreased (but not abolished) by cycloheximide (Table 1Go). No additive effects were observed when cells were exposed to both PRL and insulin or IGF-I or IGF-II (data not shown).



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 2. Iodide uptake by MCF-7 cells after treatment with different concentrations of PRL. MCF-7 human breast cells were grown as described in Materials and Methods, and then exposed to three different concentrations of PRL. Data are expressed as the mean ± SD of values obtained from at least two different experiments. Bars marked with asterisks represent significant increases over basal (unstimulated) uptake: *, P < 0.05; **, P ≤ 0.005.

 
Because hormonally induced iodide transport in thyroid cells is mediated primarily by activation of the cAMP pathway, we also evaluated uptake by MCF-7 cells after treatment with 10 µM forskolin or 1 mM (Bu)2-cAMP. As shown in Table 2Go, these cAMP-activators produced a low but significant effect on iodide uptake. Similar results were observed when cells were preincubated with the protein kinase C activator, TPA (200 mM) (Table 2Go). Treatment of MCF-7 cells with 17-ß-estradiol (10–6, 10–7, or 10–8 M) or oxytocin (10, 100, or 1000 nM) did not increase the iodide uptake (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Effects of forskolin (FSK), (Bu)2-cAMP, and TPA on iodide uptake in MCF-7 cells

 
Effects of insulin, IGF-I, IGF-II, and PRL on NIS mRNA and protein levels in MCF-7 cells

As shown in Fig. 3Go, NIS mRNA levels in MCF-7 cells were significantly increased after stimulation with insulin, IGF-I, IGF-II (Fig. 3AGo), or PRL (Fig. 3BGo). In all four cases, maximum stimulation was observed after 6 h of exposure (P = 0.002, P = 0.003, P = 0.003, and P < 0.001, respectively). Western blot analysis revealed low-level expression of the NIS protein in MCF-7 cells in the absence of insulin, IGF-I, IGF-II, or PRL. After 6 h of stimulation with each of these hormones, however, NIS protein levels were appreciably increased, and the maximum effect was observed after 12 h (Fig. 4Go).



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 3. Effects of insulin, IGF-I, IGF-II (A), and PRL (B) on NIS mRNA levels in MCF-7 cells. MCF-7 human breast cells were grown as described in Materials and Methods, and then exposed for 24 h to 10–8 M of insulin, IGF-I, or IGF-II, or 500 ng/ml of PRL. At the time points indicated in the figure, total RNA was extracted from duplicate of cells and quantified by real-time PCR. All values were normalized to glyceraldehyde-3-phosphate dehydrogenase and ß-actin controls. Data are expressed as the mean ± SD of values obtained from at least three different experiments. Bars marked with asterisks represent significant increases over basal (unstimulated) expression: *, P < 0.05; **, P < 0.003.

 


View larger version (29K):
[in this window]
[in a new window]
  		FIG. 4. Effects of IGF-I, IGF-II, insulin, and PRL on NIS protein expression in MCF-7 cells. Western blot analysis was performed as described in Materials and Methods. A, An autoradiograph of a representative experiment. B, NIS staining intensity is expressed as the mean ± SD of values obtained from two different experiments. Values marked with asterisks represent significant increases over basal (unstimulated) expression: *, P < 0.05.

 
Immunolocalization of NIS

Immunocytochemistry analysis revealed NIS protein in the plasma membranes of MCF-7 cells under basal conditions. When cells were exposed to insulin, IGF-I, or IGF-II for 12 or 24 h, NIS staining intensified in the plasma membrane and was also observed in the cytoplasm (Fig. 5Go).



View larger version (78K):
[in this window]
[in a new window]
  		FIG. 5. Immunofluorescent localization of NIS in MCF-7 cells. MCF-7 cells were analyzed by immunofluorescence microscopy, using rabbit polyclonal anti-NIS and antirabbit fluorescein isothiocyanate secondary antibody. A, NIS (green) localization in untreated MCF-7 cells (basal conditions). B, Negative control with blue that indicates 4',6-diamidino-2-phenylindole nuclear staining. NIS (green) localization after 12 h (C) and 24 h (D) of insulin stimulation (10–8 M).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ability of thyroid follicle cells to accumulate iodide has been exploited for the detection and destruction of certain types of thyroid cancer with radioiodine. If this property, which is mediated by the NIS, could be transferred to other types of cells, similar methods might be developed for the targeted treatment of various malignant tumors. In tissues that do not normally express NIS, iodide transport and organification can be induced with NIS-based gene therapy, using viral vectors (26). Endogenous NIS activity has also been documented in several nonthyroid tissues, and in these cases, hormonal or pharmacological agents could be used to enhance the expression and/or function of the protein. The latter approach is particularly interesting in light of recent reports of functional NIS expression by most human breast cancer cells (16, 17). The molecular mechanisms that regulate this expression have yet to be defined, and resolution of this issue is fundamental to any future attempts to develop targeted radioiodide therapy for breast cancer.

Previous studies have demonstrated that the NIS is the specific iodide carrier in the thyroid and also the major mediator of the gland’s regulation of TSH (7). In our present study, iodide uptake in a well-characterized model of human breast cancer, the MCF-7 cell line, was abolished by perchlorate, indicating that this activity also depends specifically on NIS-mediated transport. Our findings demonstrate that NIS expression in human breast cancer cells is also hormonally regulated. Insulin and IGFs I and II, all of which have been shown to play receptor-mediated roles in mammary cell proliferation (27, 28, 29, 30), produced parallel increases in both NIS mRNA and protein levels and significantly enhanced iodide uptake in MCF-7 cells. However, the latter effect was quantitatively inferior to that observed in normal thyroid cells stimulated with TSH, and iodide retention time in the breast cancer cells is probably very short.

Whereas NIS represents a major player in hormonally regulated iodide transport in both thyroid and mammary cells, its expression in these two tissues differs in certain respects. In thyrocytes, NIS activity is up-regulated mainly through activation of the cAMP pathway (31, 32), which also mediates the modulatory effects of human chorionic gonadotropin on NIS expression by placental cells (22). In the human breast-cancer MCF-7 cell line, the increased iodide uptake observed after cAMP activation by forskolin or (Bu)2-cAMP was statistically significant, but it was much less marked than that induced by stimulation with PRL, insulin, IGF-I, or IGF-II. This difference may reflect the existence of tissue-specific control of gene transcription involving different signal transduction pathways. Indeed, cAMP activation has also been shown to inhibit PRL-stimulated iodide uptake by normal murine mammary cells (33). An alternative explanation is that the network of transcriptional regulators in normal breast cells is altered in neoplastic breast cells as a result of a dedifferentiation process. This conclusion is consistent with previous reports showing that, whereas retinoids stimulate iodide uptake and NIS expression in both human (34) and mouse (35) breast cancer cells, retinoids have no such effect in normal human or murine mammary cells. It is important to recall that the sequence of the regulatory region of the NIS gene is not the sole determinant of iodide transport in tumor cells; in fact, in several types of thyroid tumor cells, the ability to concentrate radioiodine is compromised by alterations in the posttranscriptional steps of NIS expression (8, 9, 10, 11, 36). In addition, cis-elements in the regulatory region of the NIS gene are activated in different ways, depending on the transcription factors expressed by the specific cell type. Some of these factors that are present in thyrocytes (PAX8, for example) are not expressed in mammary cells (37), whereas others (e.g. Nkx-2.5) described in breast cells are absent in thyroid cells (38).

Several studies have demonstrated that mammary-gland radioiodide uptake activity is maximal during active lactation. For this reason, hormones like PRL and oxytocin have been used by various groups in an attempt to induce NIS expression in breast tissue. Rillema and Yu (39) and Rillema et al. (40) found that PRL stimulated iodide uptake in cultured mammary gland explants from midpregnant mice, whereas Cho et al. (41) reported that radioiodide uptake and NIS expression in histocultured human breast tumors are also modulated (in part) by PRL, which produces dose-dependent increases in the NIS mRNA levels. Our findings demonstrate that PRL dose-dependently stimulates iodide trapping and the expression of NIS in the MCF-7 human breast cancer cell line, which is known to express PRL receptors (42, 43, 44). These results appear to be in conflict with the previous observations of Kogai et al. (34), who reported that PRL has no effects on iodide uptake or NIS expression in MCF-7 cells, but this apparent discrepancy could easily be the result of the markedly different experimental conditions used in the two studies, including those involving serum concentrations in the culture medium and the durations of cell-starvation and hormonal-stimulation intervals.

It is noteworthy that none of the stimulatory effects exerted by the four hormones we tested was fully abolished by cycloheximide-induced inhibition of protein synthesis. Therefore, it seems likely that the regulatory effects of these hormones on breast cancer cell iodide uptake include a component that does not depend on the de novo synthesis of NIS protein. The same characteristic has been reported for TSH-regulation of iodide traffic in thyroid cells (45).

In conclusion, our data indicate that iodide uptake by human breast cancer cells is NIS-mediated and can be up-regulated by insulin, IGF-I, IGF-II, and PRL. The signal-transduction pathways involved in these effects are different from those that mediate hormonal regulation of iodide transport in thyroid cells. These findings provide new insights into the characterization of the molecular mechanisms that regulate the iodide-concentrating activity that has recently been observed in most breast cancer cells. Greater understanding of this regulatory network is an essential first step toward the future development of radioiodide-based treatment regimens for the management of human breast cancer.


   Acknowledgments
 
We thank M. Kent for editorial assistance.


   Footnotes
 
This work was supported by a Ministero dell’Università e della Ricerca Scientifica e Tecnologica Grant (COFIN 2003) (to D.R.), a Grant of Ministero della Salute (to S.F.), COFIN 2004 (to S.F.), and AIRC 2004 (to S.F.). I.P. was supported by a fellowship from AIRC/FIRC.

First Published Online December 28, 2004

1 F.A. and E.F. contributed equally to this article. Back

Abbreviations: (Bu)2-cAMP, Dibutyryl cAMP; FBS, fetal bovine serum; HBSS, Hanks’ balanced salt solution; NIS, sodium/iodide symporter; PBS/milk, Dulbecco’s PBS/5% nonfat dry milk; PRL, prolactin; RT, room temperature; TPA, 12-O-tetradecanoyl phorbol 13-acetate.

Received August 10, 2004.

Accepted December 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mazzaferri EL 1996 Carcinoma of follicular epithelium: radioiodine and other treatment outcomes. In: Braverman LE, Utiger RD, eds. The thyroid: a fundamental and clinical text. Philadelphia: Lippincott-Raven; 922–945
  2. Dai G, Levy O, Carrasco N 1996 Cloning and characterization of the thyroid iodide transporter. Nature 379:458–460[CrossRef][Medline]
  3. Smanik PA, Liu Q, Furminger TL, Ryu K, Xing S, Mazzaferri EL, Jhiang SM 1996 Cloning of the human sodium iodide symporter. Biochem Biophys Res Commun 226:339–345[CrossRef][Medline]
  4. Arturi F, Russo D, Schlumberger M, du Villard JA, Caillou B, Vigneri P, Wicker R, Chiefari E, Suarez HG, Filetti S 1998 Iodide symporter gene expression in human thyroid tumors. J Clin Endocrinol Metab 83:2493–2496[Abstract/Free Full Text]
  5. Lazar V, Bidart JM, Caillou B, Mahe C, Lacroix L, Filetti S, Schlumberger M 1999 Expression of the Na+/I– symporter gene in human thyroid tumors: a comparison study with other thyroid-specific genes. J Clin Endocrinol Metab 84:3228–3234[Abstract/Free Full Text]
  6. Arturi F, Russo D, Giuffrida D, Schlumberger M, Filetti S 2000 Sodium-iodide symporter (NIS) gene expression in lymph-node metastases of papillary thyroid carcinoma. Eur J Endocrinol 143:623–627[Abstract]
  7. Arturi F, Russo D, Bidart J-M, Scarpelli D, Schlumberger M, Filetti S 2001 Expression pattern of the pendrin and sodium-iodide symporter gene in human thyroid carcinoma cell lines and human thyroid tumors. Eur J Endocrinol 145:129–135[Abstract]
  8. Shen DHJ, Kloos RT, Mazzaferri EL, Jhiang SM 2001 Sodium/iodide symporter in health and disease. Thyroid 11:415–425[CrossRef][Medline]
  9. Spitzweg C, Harrington KJ, Pinke LA, Vile RG, Morris JC 2001 The sodium/iodide symporter and its potential role in cancer therapy. J Clin Endocrinol Metab 86:3327–3335[Free Full Text]
  10. Dohan O, Carrasco N 2003 Advances in Na(+)/I(–) symporter (NIS) research in the thyroid and beyond. Mol Cell Endocrinol 213:59–70[CrossRef][Medline]
  11. Filetti S, Bidart J-M, Arturi F, Caillou B, Russo D, Schlumberger M 1999 Sodium/iodide symporter: a key transport system in thyroid cancer cell metabolism. Eur J Endocrinol 141:443–457[Abstract]
  12. Dohan O, De La Vieja A, Paroder V, Riedel C, Artani M, Reed M, Ginter CS, Carrasco N 2003 The sodium/iodide symporter (NIS): characterization, regulation, and medical significance. Endocr Rev 24:48–77[Abstract/Free Full Text]
  13. Bruno R, Giannasio P, Ronga G, Baudin E, Travagli JP, Russo D, Filetti S, Schlumberger M, Sodium iodide symporter expression and radioiodine distribution in extra-thyroidal tissues. J Endocrinol Invest, in press
  14. Ajjan RA, Kamaruddin NA, Crisp M, Watson PF, Ludgate M, Weetman AP 1998 Regulation and tissue distribution of the human sodium iodide symporter gene. Clin Endocrinol (Oxf) 49:517–523[CrossRef][Medline]
  15. Spitzweg C, Joba W, Eisenmenger W, Heufelder AE 1998 Analysis of human sodium iodide symporter gene expression in extrathyroidal tissues and cloning of its complementary deoxyribonucleic acids from salivary gland, mammary gland, and gastric mucosa. J Clin Endocrinol Metab 83:1746–1751[Abstract/Free Full Text]
  16. Kilbane MT, Ajjan RA, Weetman AP, Dwyer R, McDermott EW, O’Higgins NJ, Smyth PP 2000 Tissue iodine content and serum-mediated 125I uptake-blocking activity in breast cancer. J Clin Endocrinol Metab 85:1245–1250[Abstract/Free Full Text]
  17. Tazebay UH, Wapnir IL, Levy O, Dohan O, Zuckier LS, Zhao QH, Deng HF, Amenta PS, Fineberg S, Pestell RG, Carrasco N 2000 The mammary gland iodide transporter is expressed during lactation and in breast cancer. Nat Med 6:871–888[CrossRef][Medline]
  18. Wapnir IL, van de Rijn M, Nowels K, Amenta PS, Walton K, Montgomery K, Greco RS, Dohan O, Carrasco N 2003 Immunohistochemical profile of the sodium/iodide symporter in thyroid, breast, and other carcinomas using high density tissue microarrays and conventional sections. J Clin Endocrinol Metab 88:1880–1888[Abstract/Free Full Text]
  19. Nakamoto Y, Saga T, Misaki T, Kobayashi H, Sato N, Ishimori T, Kosugi S, Sakahara H, Konishi J 2000 Establishment and characterization of a breast cancer cell line expressing Na+/I– symporters for radioiodide concentrator gene therapy. J Nucl Med 41:1898–1904[Abstract/Free Full Text]
  20. Zuckier LS, Dadachova E, Dohan O, Carrasco N 2001 The endogenous mammary gland Na(+)/I(–) symporter may mediate effective radioiodide therapy in breast cancer. J Nucl Med 42:987–988[Free Full Text]
  21. Arturi F, Presta I, Scarpelli D, Bidart JM, Schlumberger M, Filetti S, Russo D 2002 Stimulation of iodide uptake by human chorionic gonadotropin in FRTL-5 cells: effects on sodium/iodide symporter gene and protein expression. Eur J Endocrinol 147:655–661[Abstract]
  22. Arturi F, Lacroix L, Presta I, Scarpelli D, Caillou B, Schlumberger M, Russo D, Bidart JM, Filetti S 2002 Regulation by human chorionic gonadotropin of sodium/iodide symporter gene expression in the JAr human choriocarcinoma cell line. Endocrinology 143:2216–2222[Abstract/Free Full Text]
  23. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  24. Russo D, Bulotta S, Bruno R, Arturi F, Giannasio P, Derwahl M, Bidart JM, Schlumberger M, Filetti S 2001 Sodium/iodide symporter (NIS) and pendrin are expressed differently in hot and cold nodules of thyroid toxic multinodular goiter. Eur J Endocrinol 145:591–597[Abstract]
  25. Caillou B, Troalen F, Baudin E, Talbot M, Filetti S, Schlumberger M, Bidart JM 1998 Na+/I– symporter distribution in human thyroid tissues: an immunohistochemical study. J Clin Endocrinol Metab 83:4102–4106[Abstract/Free Full Text]
  26. Boland A, Magnon C, Filetti S, Bidart JM, Schlumberger M, Yeh P, Perricaudet M 2002 Transposition of the thyroid iodide uptake and organification system in nonthyroid tumor cells by adenoviral vector-mediated gene transfers. Thyroid 12:19–26[CrossRef][Medline]
  27. Strauss DS 1984 Growth-stimulatory actions of insulin in vitro and in vivo. Endocr Rev 5:356–369
  28. Khandwala HM, McCutcheon IE, Flyvbjerg A, Friend KE 2000 The effects of insulin-like growth factors on tumorigenesis and neoplastic growth. Endocr Rev 21:215–244[Abstract/Free Full Text]
  29. Papa V, Pezzino V, Costantino A, Belfiore A, Giuffrida D, Frittitta L, Vannelli GB, Brand R, Goldfine ID, Vigneri R 1990 Elevated insulin receptor content in human breast cancer. J Clin Invest 85:1503–1510
  30. Milazzo G, Giorgino F, Damante G, Sung C, Stampfer MR, Vigneri R, Goldfine ID, Belfiore A 1992 Insulin receptor expression and function in human breast cancer cell lines. Cancer Res 52:3924–3930[Abstract/Free Full Text]
  31. Trapasso F, Iuliano R, Chiefari E, Arturi F, Stella A, Filetti S, Fusco A, Russo D 1999 Iodide symporter gene expression in normal and transformed rat thyroid cells. Eur J Endocrinol 140:447–451[Abstract]
  32. Garcia B, Santisteban P 2002 PI3K is involved in the IGF-I inhibition of TSH-induced sodium/iodide symporter gene expression. Mol Endocrinol 16:342–352[Abstract/Free Full Text]
  33. Rillema JA, Marting C 1998 Cyclic AMP impairs the PRL stimulation of iodide uptake into mouse mammary tissue. Proc Soc Exp Biol Med 219:37–40[Abstract]
  34. Kogai T, Schultz JJ, Johnson LS, Huang M, Brent GA 2000 Retinoic acid induces sodium/iodide symporter gene expression and radioiodide uptake in the MCF-7 breast cancer cell line. Proc Natl Acad Sci USA 97:8519–8524[Abstract/Free Full Text]
  35. Kogai T, Kanamoto Y, Che LH, Taki K, Moatamed F, Schultz JJ, Brent GA 2004 Systemic retinoic acid treatment induces sodium/iodide symporter expression and radioiodide uptake in mouse breast cancer models. Cancer Res 64:415–422[Abstract/Free Full Text]
  36. Trouttet-Masson S, Selmi-Ruby S, Bernier-Valentin F, Porra V, Berger-Dutrieux N, Decaussin M, Peix JL, Perrin A, Bournaud C, Orgiazzi J, Borson-Chazot F, Franc B, Rousset B 2004 Evidence for transcriptional and posttranscriptional alterations of the sodium/iodide symporter expression in hypofunctioning benign and malignant thyroid tumors. Am J Pathol 165:25–34[Abstract/Free Full Text]
  37. Taki K, Kogai T, Kanamoto Y, Hershman JM, Brent GA 2002 A thyroid-specific far-upstream enhancer in the human sodium/iodide symporter gene requires Pax-8 binding and cyclic adenosine 3',5'-monophosphate response element-like sequence binding proteins for full activity and is differentially regulated in normal and thyroid cancer cells. Mol Endocrinol 16:2266–2282[Abstract/Free Full Text]
  38. Dentice M, Luongo C, Elefante A, Romino R, Ambrosio R, Vitale M, Rossi G, Fenzi G, Salvatore D 2004 Transcription factor Nkx-2.5 induces sodium/iodide symporter gene expression and participates in retinoic acid- and lactation-induced transcription in mammary cells. Mol Cell Biol 24:7863–7877[Abstract/Free Full Text]
  39. Rillema JA, Yu TX 1996 Prolactin stimulation of iodide uptake into mouse mammary gland explants. Am J Physiol 271:E879–E882
  40. Rillema JA, Yu TX, Jhiang SM 2000 Effect of prolactin on sodium iodide symporter expression in mouse mammary gland explants. Am J Physiol 279:E769–E777
  41. Cho JY, Leveille R, Kao R, Rousset B, Parlow AF, Burak Jr WE, Mazzaferri EL, Jhiang SM 2000 Hormonal regulation of radioiodide uptake activity and Na+/I– symporter expression in mammary glands. J Clin Endocrinol Metab 85:2936–2943[Abstract/Free Full Text]
  42. Shiu RP 1979 Prolactin receptors in human breast cancer cells in long-term tissue culture. Cancer Res 39:4381–4386[Abstract/Free Full Text]
  43. Reynolds C, Montone KT, Powell CM, Tomaszewski JE, Clevenger CV 1997 Expression of prolactin and its receptor in human breast carcinoma. Endocrinology 138:5555–5560[Abstract/Free Full Text]
  44. Touraine P, Martini JF, Zafrani B, Durand JC, Labaille F, Malet C, Nicolas A, Trivin C, Postel-Vinay MC, Kuttenn F, Kelly PA 1998 Increased expression of prolactin receptor gene assessed by quantitative polymerase chain reaction in human breast tumors versus normal breast tissues. J Clin Endocrinol Metab 83:667–674[Abstract/Free Full Text]
  45. Riedel C, Levy O, Carrasco N 2001 Post-transcriptional regulation of the sodium/iodide symporter by thyrotropin. J Biol Chem 276:21458–21463[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
T. Kogai, E. Ohashi, M. S. Jacobs, S. Sajid-Crockett, M. L. Fisher, Y. Kanamoto, and G. A. Brent
Retinoic Acid Stimulation of the Sodium/Iodide Symporter in MCF-7 Breast Cancer Cells Is Meditated by the Insulin Growth Factor-I/Phosphatidylinositol 3-Kinase and p38 Mitogen-Activated Protein Kinase Signaling Pathways
J. Clin. Endocrinol. Metab., May 1, 2008; 93(5): 1884 - 1892.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
T Kogai, K Taki, and G A Brent
Enhancement of sodium/iodide symporter expression in thyroid and breast cancer.
Endocr. Relat. Cancer, September 1, 2006; 13(3): 797 - 826.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
O. Dohan, A. De la Vieja, and N. Carrasco
Hydrocortisone and Purinergic Signaling Stimulate Sodium/Iodide Symporter (NIS)-Mediated Iodide Transport in Breast Cancer Cells
Mol. Endocrinol., May 1, 2006; 20(5): 1121 - 1137.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
C. Puppin, F. D'Aurizio, A. V. D'Elia, L. Cesaratto, G. Tell, D. Russo, S. Filetti, E. Ferretti, E. Tosi, T. Mattei, et al.
Effects of Histone Acetylation on Sodium Iodide Symporter Promoter and Expression of Thyroid-Specific Transcription Factors
Endocrinology, September 1, 2005; 146(9): 3967 - 3974.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
90/4/2321    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arturi, F.
Right arrow Articles by Filetti, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arturi, F.
Right arrow Articles by Filetti, S.
Related Collections
Right arrow Endocrine Oncology
Right arrow Thyroid

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals