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Endocrinology Vol. 145, No. 11 5397-5405
Copyright © 2004 by The Endocrine Society

Adenovirus-Mediated Transfer of Thyroid Transcription Factor-1 Induces Radioiodide Organification and Retention in Thyroid Cancer Cells

Fumihiko Furuya, Hiroki Shimura, Asako Miyazaki, Katsumi Taki, Kazuyasu Ohta, Kazutaka Haraguchi, Toshimasa Onaya, Toyoshi Endo and Tetsuro Kobayashi

Third Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Yamanashi 409-3898, Japan

Address all correspondence and requests for reprints to: Dr. Tetsuro Kobayashi, Third Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Tamaho, Yamanashi 409-3898, Japan. E-mail: tetsurou{at}yamanashi.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Loss of thyroid-specific gene expression and functions accompanied by loss of thyroid transcription factors render them unresponsive to radioiodide therapy in poorly differentiated and anaplastic thyroid cancer. In anticipation of reactivation of thyroid functions, we investigated the effect of thyroid transcription factor-1 (TTF-1) gene transfer on thyroid cancer cells. Reexpression of thyroperoxidase (TPO) and thyroglobulin (Tg) mRNA and protein was detected in poorly differentiated human thyroid cancer cells that were infected with an adenovirus vector containing TTF-1 (AdTTF-1). Although TTF-1 gene transfer faintly induced iodide uptake, the induction of sodium/iodide symporter (NIS) mRNA was not observed in AdTTF-1-infected cells. To analyze the effect of TTF-1 on iodide metabolism, we transfected an NIS expression vector into BHP18–21v cells and cloned a cell line (N-BHP18–21v) that stably expressed NIS. The treatment of N-BHP18–21v cells with AdTTF-1 significantly increased the amount of protein-bound radioiodide and prolonged the iodide efflux. AdTTF-1 injections significantly induced iodide retention and organification in tumors formed from N-BHP18–21v cells in nude mice. These results indicate that AdTTF-1 specifically induces iodide organification and retards iodide efflux in thyroid cancer cells in vitro and in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A UNIQUE PROPERTY of thyroid follicular cells is the ability to trap and concentrate iodide, which depends on expression of the sodium/iodide symporter (NIS), thyroglobulin (Tg), and thyroperoxidase (TPO) (1). Because of its capacity for iodide trapping and concentration, radioiodide is an effective therapy in the treatment of differentiated thyroid carcinomas (2, 3, 4). However, 33% of metastatic thyroid cancer showed no radioiodide uptake (5). In addition, anaplastic thyroid carcinoma, which shows no radioiodide accumulation, is highly malignant, with a median survival of 2–6 months (6, 7). Because no effective therapy is available for patients with these poorly differentiated thyroid carcinoma, the development of novel therapeutic strategies, including gene therapy, is an urgent priority.

NIS protein mediates iodide uptake in normal and well differentiated neoplastic thyroid cells. TPO iodinates Tg, which leads to iodide retention within thyroid follicles. Concentration of these thyroid-specific functions suggests that restoration of NIS, TPO, and Tg expression could provide a basis for radioiodide treatment of thyroid carcinomas. The loss of thyroid-specific functions associated with dedifferentiation in poorly differentiated and anaplastic thyroid cancer cells ordinarily render them unresponsive to radioiodide therapy. We reported that Na131I administration did not decrease the volumes of experimental tumors formed by malignantly transformed rat thyroid cells Tc-rNIS that were stably transfected with rat NIS cDNA, but possessed no TPO or Tg expression (8). Concomitant reexpression of NIS, TPO, and Tg, therefore, would seem necessary for the iodide uptake and retention that could permit effective radioiodide therapy. Recently, the importance of coexpression of NIS and TPO in intracellular radioiodide retention was shown in cultured lung cancer cells (9).

The expression of these genes is controlled by the interaction of a complement of thyroid-specific transcription factors with the respective promoters of these genes (10, 11). Three transcription factors have been cloned, thyroid transcription factor-1 (TTF-1), TTF-2, and Pax-8 (11, 12, 13). TTF-1 is a homeodomain-containing, DNA-binding protein expressed in thyroid and lung (12). TTF-1 plays a decisive role in the determination and maintenance of cellular phenotype by activating TPO and Tg gene transcription (11, 12, 14, 15, 16). Additionally, we have reported that TTF-1 activates the rat NIS promoter by a direct interaction with its proximal enhancer region in the rat NIS gene (17).

The expression of thyroid-specific genes and their transcription factors is lost in cultured thyroid cancer cells derived from follicular, papillary, and anaplastic human thyroid carcinoma (18). Ross et al. (18) reported that transfection with an expression vector for TTF-1 resulted in the stimulation of transcription to a different extent for TPO and Tg promoter. Furthermore, we reported that cotransfection of a plasmid containing the rat Tg promoter and the luciferase gene with the TTF-1 expression plasmid resulted in activation of the Tg promoter in thyroid cancer cells, but not in nonthyroid cells (19). Herein, to efficiently induce reactivation of endogenous thyroid-specific genes, we constructed an adenovirus vector (AdTTF-1) for TTF-1 gene transfer.

In the present study, to develop a way to sensitize poorly differentiated thyroid carcinoma to radioiodide therapy, we investigated reexpression of thyroid-specific genes, NIS, TPO, and Tg, induced by AdTTF-1. We then examined radioiodide uptake and organification to confirm the function of the reexpressed, thyroid-specific genes in vitro or in vivo. These results suggested that TTF-1 is important in the restoration of thyroid specific-gene expression in thyroid cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
BHP18–21 and BHP7–13 cells, derived from primary tumors with histological features of human papillary thyroid cancer, were provided by Dr. J. M. Hershman (Endocrine Research Laboratory, West Los Angeles Veterans Affairs Medical Center, Los Angeles, CA) and maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) (20). BHP18–21v cells expressing Pax-8, but neither TTF-1 nor Tg genes, were isolated from BHP18–21 cells (19, 21). BHP7–13 cell line expresses Pax-8, but TTF-1 and Tg were not detected in Northern blotting analyses (20). A human anaplastic thyroid carcinoma cell line, ARO (22), which lacks Pax-8, Tg, and NIS, was donated by Dr. H. Namba (Atomic Bomb Disease Institute, Nagasaki University School of Medicine, Nagasaki, Japan) and incubated with RPMI 1640 containing 10% FCS. HepG2, a human hepatocellular cancer cell line, was purchased from American Type Culture Collection (Manassas, VA) and cultured in DMEM supplemented with 10% FCS. HepG2 cells do not express TTF-1 or Pax-8 (19). Rat NIS cDNA was introduced into Chinese hamster ovary cells (CHO-K1 cells). The stably transfected cells (CHO-rNIS cells), which have been previously described (17), were used as a negative control in AdTTF-1-mediated iodide accumulation assays.

Stable transfection of human NIS expression vector in BHP18–21v cells
Human NIS cDNA (23) was subcloned into the eukaryotic expression vector pcDNA3.1zeo (Invitrogen Life Technologies, Carlsbad, CA) and introduced into BHP18–21v cells by electroporation. Stably transfected cells were selected using zeocin (Invitrogen Life Technologies) and cloned by limited dilution. The cloned cell line N-BHP18–21v, which exhibited the highest iodide uptake activity and immunoreactive NIS protein expression, was used for the polyclonal antibody (24).

Construction of the recombinant adenoviral vectors
AdTTF-1 is a {Delta}E1-{Delta}E3 recombinant adenovirus expressing the rat TTF-1 gene under the control of the immediate early promoter of cytomegalovirus (CMV). Construction of the AdTTF-1 virus has been previously described (19). AdLacZ, which contains the CMV promoter-controlled lacZ gene, was provided by Quantum Biotechnologies (Montréal, Canada) and used as a control. Recombinant adenoviruses were plaque-purified, harvested 48 h after infection of 293 cells, and purified by double cesium chloride gradient ultracentrifugation (25). Viral titers were determined by plaque assays using cultured 293 cells (19).

Northern blot analysis
Total RNA was isolated from cultured cells using the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Northern blot analysis was performed as previously described (26). Human TPO cDNA (27) and human Tg cDNA (28) were obtained by RT-PCR from human thyroid mRNA. All probes were radiolabeled using a random primer labeling kit (Takara, Kyoto, Japan).

Immunoperoxidase staining
Immunostaining of thyroid cancer cells infected with adenoviral vectors was performed to confirm the expression of TPO and Tg proteins in response to AdTTF-1. After infection with adenovirus, cells were fixed for 15 min in –10 C methanol, air-dried, and washed three times with PBS. The fixed cells were incubated with anti-Tg (NeoMarkers, Fremont, CA) or anti-TPO monoclonal antibody (HyTest Ltd., Turku, Finland) for 30 min and washed three times with PBS. Cells were incubated with biotinylated secondary antibody, washed, and exposed to avidin-biotinylated horseradish peroxidase enzyme reagent and peroxidase substrate (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Then nuclei were stained with Mayer’s hematoxylin (Wako Biochemicals, Osaka, Japan). Cells were examined for staining after rinsing with H2O.

Radioiodide uptake assay in vitro
Cells were grown in 12-well plates, washed with Hanks’ balanced salt solution (HBSS), and incubated for 1 h at 37 C with 500 µl HBSS containing 0.1 µCi carrier-free Na125I (Amersham Biosciences, Piscataway, NJ) and 10 µM NaI, with or without 300 µM NaClO4 (23). The medium containing 125I was removed, and the cells were washed twice with 1 ml HBSS. Cell-associated radioactivity was measured with a {gamma}-counter after extraction with 0.1 N NaOH.

Radioiodide organification assay in vitro
N-BHP18–21v cells were infected with adenovirus for 48 h. Methimazole (MMI; 500 µM; Sigma-Aldrich Corp., St. Louis, MO), a TPO-specific inhibitor, was added 24 h before the assay. Adenovirus-infected N-BHP18–21v cells were then exposed to 125I (0.1 µCi/well in six-well plates) in HBSS containing 10 µM NaI with or without 0.1 mM H2O2 at 37 C for 1 h. Medium containing 125I was removed, and cells were washed twice with fresh nonradioactive medium. Cell lysates were prepared with 0.5 ml 0.1 N NaOH, and the radioiodide accumulation of the cells was measured with a {gamma}-counter. Proteins in the cell lysates were precipitated by the addition of 0.5 ml 40% trichloroacetic acid (TCA; final concentration, 20%). Precipitated proteins were collected by centrifugation at 3300 x g for 30 min and were washed twice with nonradioactive medium. Radioactivity in the pellets was measured with a {gamma}-counter.

Radioiodide efflux assay in vitro
N-BHP18–21v cells were cultured in 12-well plates. The cells were infected with adenovirus for 24 h and incubated with nonradioactive medium for 24 h. Adenovirus-infected N-BHP18–21v cells were exposed to 125I (0.1 µCi/well in 12-well plates) in HBSS containing 10 µM NaI at 37 C for 1 h. Medium was replaced with nonradioactive medium every 5 min, and the release of radioactivity into the medium was monitored using a {gamma}-counter (29). Finally, cells were lysed with 1 ml 0.1 N NaOH, and the residual radioiodide was measured with a {gamma}-counter. Total radioactivity present at the initiation of the efflux assay was calculated by adding the counts in the final cell extract to the medium counts. The approximation curve was determined using Excel software (Microsoft Corp., Redmond, WA), and the t1/2 value was calculated.

Analysis of iodide accumulation and organification in vivo
Ten million BHP18–21v or N-BHP18–21v cells were transplanted in the abdominal sc tissue of 8-wk-old male nude mice (BALB/c {nu}/{nu} mice). These mice were fed a low iodide diet (Oriental Yeast, Tokyo, Japan) and were given 100 µg/kg·d L-T4 (Wako Biochemicals) in their drinking water before the formation of tumor (8). After 3 wk, the tumors reached approximately 1 cm in diameter, and 5 x 108 plaque-forming units AdTTF-1 or AdLacZ in 100 µl PBS were injected into the tumors for 4 d. Na125I (10 µCi; Amersham Biosciences) in PBS was injected ip. Three, 12, or 48 h later, the mice were killed, and radioactivity was analyzed using a BAS2500 image analyzer (Fuji Film Corp., Tokyo, Japan). The mice were exposed to the imaging plates for 3 min. To investigate the ratio of tumor or stomach radioactivity to liver radioactivity, the tumors and organs were removed, and their weights and radioactivity were measured.

Forty-eight hours after the injection of Na125I, tumors were removed. Parts of the tumors were homogenized. After preparation of tissue lysate with 300 µl 1 N NaOH, insoluble materials were removed from the lysate by centrifugation at 3300 x g for 10 min at 4 C. Proteins in the tissue lysates were precipitated by the addition of 600 µl 40% TCA. The precipitated proteins were collected by centrifugation at 3300 x g for 20 min and washed twice. Radioactivity in the pellets was measured with a {gamma}-counter. Soluble protein was quantified by the Bradford method using the RCDC protein assay (Bio-Rad Laboratories, Hercules, CA).

All experiments with mice were performed under approved protocols from the institutional animal care and use committee and the radiation safety committee at University of Yamanashi.

Statistical analysis
Data are expressed as the mean ± SEM. Differences between groups were examined for statistical significance using a t test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AdTTF-1-induced reexpression of TPO and Tg in BHP18–21v and BHP7–13 cells
To investigate the effect of TTF-1 gene transfer on the reexpression of TPO and Tg genes, we constructed a recombinant adenovirus vector that contained the CMV promoter and rat TTF-1 cDNA, AdTTF-1 (19). BHP18–21v and BHP7–13 cells (TTF-1–/Pax-8+/TPO–/Tg–) (19) were infected with AdTTF-1 or AdLacZ control adenovirus. After 24 h of incubation with 100, 300, or 1000 multiplicity of infection (MOI) of adenoviral vectors, cells were incubated for 48 h in virus-free medium. Based on Northern blot analysis, reexpressed TPO and Tg mRNA were detected in 100, 300, and 1000 MOI AdTTF-1-infected cells, but not in AdLacZ-infected cells (Fig. 1Go, A and B). Quantification of mRNA levels showed an MOI-dependent increase in Tg and TPO mRNA levels (Fig. 1Go, A and B). In contrast, in ARO cells (TTF-1+/Pax-8–/TPO–/Tg–), reexpression of TPO and Tg was not observed even if the cells were treated with AdTTF-1 (Fig. 1CGo). We also examined the effect of AdTTF-1 on thyroid-specific gene expression in nonthyroid HepG2 cells (TTF-1–/Pax-8–/TPO–/Tg–) (19). Even 1000 MOI AdTTF-1 were unable to induce the expression of TPO and Tg mRNA (Fig. 1DGo). These results indicate that AdTTF-1 specifically affected the regulatory machinery of transcription in the expression of TPO and Tg in Pax-8-expressing thyroid cancer cells.



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  		FIG. 1. Reexpression of TPO and Tg mRNA induced by AdTTF-1. The mRNA levels of thyroid-specific genes in BHP 18–21v (TTF-1–, Pax-8+; A), BHP7–13 (TTF-1(–), Pax-8+; B), ARO (TTF-1+, Pax-8–; C), and HepG2 (TTF-1–, Pax-8–; D) cells infected with 100, 300, or 1000 MOI AdTTF-1 or AdLacZ were investigated by Northern blot analysis. Each cell line was infected with AdTTF-1 or AdLacZ for 24 h, then incubated with adenovirus-free medium for 48 h. Equal amounts of total RNA (20 µg/lane) were subjected to sequential Northern blot analysis using human TPO, human Tg, or human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes. In BHP18–21v (A) and BHP7–13 (B) cells, quantification was performed using a BAS 2000 image analyzer (Fuji Photo Film Corp.). The TPO or Tg mRNA/GAPDH mRNA ratio is expressed by arbitrarily setting the control value from samples infected with 1000 MOI AdTTF-1 to 1.

 
We also investigated time dependence of AdTTF-1-induced reexpression of TPO and Tg in BHP18–21v cells. We isolated total RNA of AdTTF-1-infected cells at 24, 48, and 72 h after starting the adenovirus infection. The amounts of TPO and Tg mRNA gradually increased and peaked 72 h after the infection with 300 MOI AdTTF-1 (Fig. 2Go).



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  		FIG. 2. Time-dependent mRNA expression of thyroid-specific genes induced by AdTTF-1. Reexpression of thyroid-specific genes in BHP 18–21v cells incubated for 24, 48, or 72 h was analyzed. Cells were incubated with medium containing 300 MOI AdTTF-1 for 24 h, then incubated with adenovirus-free medium. Equal amounts of total RNA (20 µg/lane) were subjected to sequential Northern blot analyses using human TPO, human Tg, or human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes. Quantification was performed using a BAS 2000 image analyzer (Fuji Photo Film Corp.). TPO or Tg mRNA/GAPDH mRNA ratio is expressed by arbitrarily setting the control value from samples incubated for 72 h to 1.

 
These findings indicate that AdTTF-1 specifically restored the expression of TPO and Tg genes in dedifferentiated thyroid cancer cells that expressed Pax-8.

AdTTF-1-induced reexpression of TPO and Tg proteins in BHP18–21v and BHP7–13 cells
To analyze whether AdTTF-1-up-regulated TPO or Tg mRNA is translated into protein, BHP18–21v cells were stained with anti-TPO or anti-Tg antibody. TPO protein was detected by immunocytochemistry in the cell membrane and cytosol of BHP18–21v cells infected with 300 MOI AdTTF-1 (Fig. 3Go). Uninfected cells and cells infected with 300 MOI AdLacZ were not stained by anti-TPO antibody. Immunoreactive Tg protein was stained with anti-Tg monoclonal antibody in the cytosol of AdTTF-1-infected BHP18–21v cells. Tg staining was not observed in uninfected or AdLacZ-infected cells. These results clearly show that AdTTF-1 induced the expression of TPO and Tg proteins.



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  		FIG. 3. Detection of immunoreactive TPO and Tg proteins in AdTTF-1-infected BHP18–21v cells. BHP18–21v cells were infected with 300 MOI AdTTF-1 or AdLacZ for 24 h, then incubated with adenovirus-free medium for 24 h. The cells were fixed and incubated with anti-TPO or anti-Tg monoclonal antibody. Cells were then stained with avidin-biotinylated horseradish peroxidase.

 
AdTTF-1-enhanced radioiodide accumulation in N-BHP18–21v cells in vitro
BHP18–21v cells were infected with 30, 100, or 300 MOI AdTTF-1 for 24 h, then incubated for 24 h without adenovirus. Iodide uptake was measured after incubating the cells with HBSS containing 0.1 µCi 125I for 1 h. We detected faint iodide accumulation in the AdTTF-1-infected BHP18–21v cells (Fig. 4AGo, bar 1 vs. bars 2–4). The iodide accumulation was inhibited by sodium perchlorate. However, Northern blot analysis and RT-PCR revealed that NIS mRNA was not expressed in AdTTF-1-infected cells (data not shown). These results indicate that TTF-1 might stimulate novel iodide transport or inhibit iodide efflux.



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  		FIG. 4. Radioiodide accumulation in BHP18–21v and N-BHP18–21v cells infected with AdTTF-1. A, BHP18–21v cells were infected with 30, 100, or 300 MOI AdTTF-1 for 24 h, then incubated with adenovirus-free medium for 24 h. B, N-BHP18–21v cells were infected with 30, 100, or 300 MOI AdTTF-1 for 24 h, then incubated with adenovirus-free medium for 24 h. CHO-rNIS cells were also infected with 300 MOI AdTTF-1 for 24 h, then incubated with adenovirus-free medium for 24 h. All cells were incubated in medium containing 0.1 µCi Na125I with or without 300 µM NaClO4 for 1 h. After the cells were washed twice, intracellular radioiodide was extracted by NaOH. Data are expressed as the mean ± SEM (n = 6). {diamondsuit}, P < 0.05 compared with uninfected BHP18–21v cells; *, P < 0.01, compared with uninfected N-BHP18–21v cells; {diamond}, P < 0.05 compared with NaClO4-treated cells.

 
Because the radioiodide count was too low to investigate the effect of AdTTF-1 on iodide-transporting machinery other than NIS, we established N-BHP18–21v cells, which were stably transfected with an expression vector containing human NIS cDNA. N-BHP18–21v cells exhibited a 96-fold increase in radioiodide accumulation compared with uninfected BHP18–21v cells (Fig. 4BGo, bar 1 vs. bar 2). These radioiodide uptakes were completely inhibited by sodium perchlorate. AdTTF-1 markedly enhanced radioiodide accumulation in N-BHP18–21v cells; 30, 100, and 300 MOI AdTTF-1 induced 7-, 14-, and 23-fold increases in radioiodide accumulation, respectively (Fig. 4BGo, bar 2 vs. bars 3–5).

To investigate the specificity of the TTF-1 effect on iodide uptake, we established an NIS-transfected nonthyroid cell line, CHO-rNIS (Fig. 4BGo). In contrast to N-BHP18–21v cells, infection with AdTTF-1 did not augment radioiodide accumulation in CHO-rNIS cells (Fig. 4BGo, bar 7). These results suggest that AdTTF-1 induced a factor that specifically increased the radioiodide accumulation in thyroid cancer cells.

AdTTF-1-induced radioiodide organification in N-BHP18–21v cells
Infection with AdTTF-1 induced iodide uptake in BHP18–21v cells and significantly increased radioiodide accumulation in N-BHP18–21v cells (Fig. 4Go). Because AdTTF-1 also induced the reexpression of TPO and Tg proteins (Fig. 3Go), we hypothesized that TTF-1 gene transfer induced iodide organification or inhibited iodide efflux. To analyze the mechanism of AdTTF-1-enhanced iodide accumulation, we measured AdTTF-1-induced radioiodide organification in N-BHP18–21v cells. In uninfected N-BHP18–21v cells without H2O2, 2.88 ± 0.18% of accumulated radioiodide was bound to the protein fraction (Fig. 5Go, bar 1). There were no significant differences in iodide organification between the uninfected cells treated with or without H2O2 (Fig. 5Go, bar 1 vs. bar 4). One hundred and 300 MOI AdTTF-1 significantly enhanced radioiodide organification to 5.00 ± 0.70% (P < 0.05) and 5.34 ± 0.73% (P < 0.05), respectively (Fig. 5Go, bar 1 vs. bars 2 and 3). Treatment with 0.1 mM H2O2 in cells infected with 100 and 300 MOI AdTTF-1 enhanced organification to 12.59 ± 2.02% and 7.35 ± 0.78%, respectively (Fig. 5Go, bar 2 vs. 5 and bar 3 vs. 6). Pretreatment of the cells with MMI inhibited the AdTTF-1-induced iodide organification. These results demonstrate that the TPO induced by AdTTF-1 facilitated iodide organification into protein substrates and that the iodide organification was enhanced by H2O2.



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  		FIG. 5. AdTTF-1-induced iodide organification in N-BHP18–21v cells. N-BHP18–21v cells were infected with AdTTF-1 and incubated with or without 500 µM MMI. Cells were then exposed to 0.1 µCi/well Na125I for 1 h with or without 0.1 mM H2O2. After the cells were washed twice, the radioactive uptake of 125I was counted. The radioactivity of 125I-bound protein was also determined by TCA precipitation. The percentage of protein-bound radioiodide is presented. Data are expressed as the mean ± SEM (n = 6). *, P < 0.05.

 
Although infection with 300 MOI AdTTF-1 induced higher intracellular radioiodide retention than that in the 100 MOI infection (Fig. 4BGo), the protein-bound radioiodine/total uptake ratio in H2O2-treated cells with the 300 MOI infection was lower than that in cells with the 100 MOI infection (Fig. 5Go). There was no decrease in protein-bond iodine/DNA in the cells infected with 300 MOI AdTTF-1 (2,376 ± 307 cpm/µg DNA) compared with the 100 MOI infection (2,002 ± 248 cpm/µg DNA). Moreover, the 300 MOI infection significantly increased intracellular protein-unbound iodide retention (30,529 ± 3,129 cpm/µg DNA) compared with 100 MOI (15,082 ± 3,129 cpm/µg DNA). In comparison with no infection (1,560 ± 236 cpm/µg DNA), infection with 300 MOI AdTTF-1 induced a 20-fold increase in intracellular protein-unbound radioiodine. These results demonstrate that TTF-1 gene transfer induces not only iodide organification, but also intracellular free iodide retention.

AdTTF-1 induced the prolongation of iodide efflux in N-BHP18–21v cells
To determine whether protein-unbound iodide retention could be induced in AdTTF-1-infected cells, we performed a radioiodide efflux assay (Fig. 6Go). N-BHP18–21v cells infected with 300 MOI AdTTF-1 or AdLacZ were exposed to radioiodide, and the release of radioactivity into the medium was monitored every 5 min. There was a rapid efflux of radioactivity from the AdLacZ-infected cells (t1/2 = 6.2 min), and cellular radioactivity was almost completely released into the medium over 30 min. In contrast, iodide efflux was prolonged in AdTTF-1-infected cells (t1/2 = 15.2 min). These results indicate that AdTTF-1 inhibited iodide efflux and that the increased amounts of protein-unbound radioiodide could be involved in radioiodide accumulation in AdTTF-1-infected N-BHP18–21v cells.



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  		FIG. 6. Iodide efflux assay in AdTTF-1- or AdLacZ-infected N-BHP18–21v cells. N-BHP18–21v cells infected with 300 MOI AdTTF-1 or AdLacZ were exposed to 0.1 µCi/well Na125I for 1 h. The medium was replaced with fresh nonradioactive HBSS every 5 min, and its radioactive content was measured using a {gamma}-counter. After the last medium was removed, the cells were extracted with 1 ml 0.1 N NaOH. The total radioactivity present at the initiation of the efflux study (100%) was calculated by adding the counts in the final tissue extract to the medium counts. Data are expressed as the mean ± SEM (n = 8).

 
AdTTF-1 leads to efficient iodide accumulation in vivo
To evaluate the efficiency of AdTTF-1 in vivo, 1 x 107 N-BHP18–21v cells were sc injected into the left flanks of nude mice. One week later, the cells formed small tumors at the injection sites. The tumors grew to 1 cm in diameter by 3 wk after injection. At this point, AdTTF-1 or AdLacZ was injected into the tumors (5 x 108 PFU in 100 µl PBS) for 4 d. Autoradiographic imaging of AdLacZ-treated and AdTTF-1-treated mice revealed the accumulation of radioiodide in the tumors and thyroid 3 and 12 h after the injection of Na125I. However, after 48 h, 125I accumulation in AdLacZ-treated tumors had almost disappeared. In contrast, imaging at 48 h clearly showed 125I accumulation in tumors of AdTTF-1-treated mice (Fig. 7AGo).



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  		FIG. 7. Intratumoral radioiodide accumulation induced by AdTTF-1 in vivo. N-BHP18–21v cells were transplanted into the left flank of nude mice by sc injection. Mice were treated with L-T4 to avoid massive uptake by the thyroid. AdTTF-1 or AdLacZ (5 x 108 PFU) was injected into the tumors for 4 d. After the injection of 10 µCi 125I, radioactivity in the mice was analyzed using a BAS2500 imaging analyzer. A, Typical images 3, 12, and 48 h after the injection. B, After calculating the radioiodide accumulation per tissue weight, the tumor/liver and stomach/liver radioiodide uptake ratios were determined. C, The intratumoral radioactivity of 125I bound to protein was determined by TCA precipitation. Radioactivity was measured with a {gamma}-counter and was normalized by dividing by the protein weight. Data are expressed as the mean ± SEM (n = 4–5). *, P < 0.05 compared with AdLacZ-treated tumors.

 
To examine the time-dependent accumulation of 125I in these mice, the tumor/liver and stomach/liver 125I concentration ratios were measured at 3, 12, and 48 h after injection of 10 µCi Na125I (Fig. 7BGo). At 12 and 48 h, the tumor/liver 125I concentration ratios were 15.67 ± 4.29 and 49.73 ± 5.81, respectively, in AdTTF-1-treated mice and 4.01 ± 0.71 and 6.56 ± 2.61, respectively, in AdLacZ-treated mice. There were significant differences in the tumor/liver 125I concentration ratios at 12 and 48 h between AdTTF-1-treated and AdLacZ-treated mice (P < 0.05). In the stomach, which naturally expresses NIS, the 125I concentration ratio at 12 h was increased to 23.12 ± 12.71. However, no 125I was observed in the stomach at 48 h.

To analyze iodide organification in vivo, we measured protein-bound radioiodide in xenotransplanted tumors (Fig. 7CGo). The protein-bound radioiodide level in AdTTF-1-treated mice was 208.43 ± 37.43 cpm/mg protein, whereas that in AdLacZ-treated mice was 30.65 ± 14.41 cpm/mg protein (P < 0.05). These results demonstrate that AdTTF-1 induced radioiodide accumulation in vivo at least in part via AdTTF-1-induced radioiodide organification.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Transcription factors or certain reagents have been found to induce thyroid-specific gene expression in dedifferentiated thyroid cancer cells. Fagin et al. (30) reported that papillary thyroid cancer cells transfected with wild-type p53 showed reexpression of TPO mRNA and protein via reexpression of Pax-8 mRNA. Schmutzler et al. (31) reported that all-trans-retinoic acid increased NIS mRNA in human follicular thyroid cancer cells. We also detected the histone deacetylase inhibitor (HDACI)-induced expression of NIS, TPO, and Tg using RT-PCR analysis (21). In the present report we provide the first evidence suggesting that adenovirus-mediated transfer of TTF-1 induces the simultaneous reexpression of TPO and Tg mRNA, which can be detected by Northern blot analysis, and proteins in human thyroid cancer cells. We also demonstrate that AdTTF-1-infected cancer cells that stably expressed NIS increased their radioiodide uptake and organification in vitro and in vivo.

In the present study the action of TTF-1 on Tg and TPO expression was evident in BHP18–21v, BHP7–13 (TTF-1–, Pax-8+) papillary thyroid cancer cells, but not in ARO (TTF-1+, Pax-8–) anaplastic thyroid cancer cells. Because synergistic actions of TTF-1 and Pax-8 on TPO or Tg promoters were reported (32), we speculated that the expression of both TTF-1 and Pax-8 may be required for TPO and Tg mRNA expression high enough to be detected by Northern blot analysis. These results suggest that this strategy might be relevant only to tumors with residual Pax-8 expression. In contrast, we recently reported that overexpression of TTF-1 specifically restores Tg promoter activity in ARO as well as BHP cells (19). The discrepancy between the promoter activity and mRNA expression of the Tg gene might be due to the difference in target DNA conditions between chromosomal and naked DNA. We speculated that histone acetylation might be involved in this discrepancy, because we recently reported that HDACIs induced reexpression of Tg and TPO mRNA and protein in ARO cells (21). This was associated with restoration of TTF-1 expression (21).

In the rat NIS gene, TTF-1 activates the rat NIS promoter by a direct interaction with its proximal enhancer region (17). In the human NIS gene, the TTF-1 binding site is in the NIS upstream enhancer region, but TTF-1 does not act as a trans-activating factor on the human NIS upstream element (33). Rather than TTF-1, Pax-8 was shown to be a main regulator of human NIS promoter (33). In the present study BHP18–21v cells showed no detectable NIS expression regardless of whether they were infected with AdTTF-1 (data not shown). These results indicate that TTF-1 most likely has no activity as a trans-activator on the human NIS gene in human thyroid cells. However, the fact that Pax-8-positive BHP cells do not express NIS mRNA (21) raises the possibility that NIS expression can be regulated by modifications of its chromosomal genome. Our recent report (21), which showed HDACI-induced restoration of the NIS and TTF-1 expression in BHP and ARO cells, suggests inactivation of NIS promoter by deacetylation of histone in thyroid cancer cells. Venkataraman et al. (7) also reported that demethylation of NIS promoter by 5-azacytidine restored NIS expression in cultured thyroid cancer cells. This was accompanied by TTF-1 reexpression (7). In BHP18–21v cells, treatment with 0.3–10 µM 5-azacytidine, however, failed to restore NIS mRNA expression and iodide uptake (data not shown).

AdTTF-1 induced a small, but significant, iodide accumulation, that was inhibited by sodium perchlorate in BHP18–21v cells. In normal thyroid cells, NIS mediates active iodide transport into thyroid cells (34). Electrophysiological analysis revealed that perchlorate inhibits the NIS-mediated inward current of iodide (35). Based on these findings, one may speculate that AdTTF-1 induces iodide accumulation via up-regulation of NIS expression or function. However, it was difficult to clarify this mechanism because we were unable to observe increased NIS mRNA levels. Another possibility is that TTF-1 induces an unknown protein that mediates perchlorate-sensitive iodide transport into thyroid cancer cells. Recently, Rodriguez et al. (36) characterized a putative apical iodide transporter. Because sodium perchlorate inhibited AdTTF-1-induced iodide accumulation, it is possible that AdTTF-1 might enhance the expression or function of apical iodide transporter protein.

In differentiated thyroid cells, iodide organification is formed by iodination and intermolecular coupling of specific tyrosine residues in Tg (37). TPO is the primary enzyme involved in this process (38). TPO is active at the apical pole of the thyrocytes, where oxidizing H2O2 is produced (39). Boland et al. (40) reported that iodide organification was observed in thyroid cancer cells cotransfected with NIS and TPO in the presence of exogenous H2O2. We detected increased amounts of protein-bound iodide in AdTTF-1-infected cells without the addition of H2O2 as well as H2O2-induced amplification of AdTTF-1-induced iodide organification. These results indicate that AdTTF-1 could induce reexpression of functional TPO protein and that the expression of NIS, TPO, and Tg in the presence of H2O2 induced efficient iodide organification in thyroid cancer cells. This conclusion is also supported by the observation that iodide accumulation did not occur in AdTTF-1-infected CHO-rNIS cells that did not express TPO or Tg. In BHP18–21v cells, AdTTF-1 induced a small, but significant, amount of iodide accumulation (Fig. 4Go), suggesting that the reexpressed TPO induced by TTF-1 could be involved with thyroid-specific iodide accumulation.

In the present report, 300 MOI AdTTF-1 produced a 23-fold increase in radioiodide accumulation, compared with 300 MOI AdLacZ. Otherwise, in 300 MOI AdTTF-1-infected cells, 7% of the accumulated radioiodide was bound to the protein fraction after exposure to Na125I (0.1 µCi/well) for 1 h. When the protein in AdTTF-1- or AdLacZ-treated tumors was precipitated using TCA, the radioactivity of precipitated protein was approximately 5% of the total accumulated radioiodide in AdTTF-1-treated tumors. These results suggested that AdTTF-1-induced iodide retention consists of iodide organification and retarded free iodide efflux. There is a possibility that AdTTF-1 induces the intracellular accumulation of 125I in thyroid cancer cells via down-regulation of some transporter of iodine responsible for the iodide efflux from thyrocytes.

Radioiodide has been recently proposed as a valuable tool in treatment of nonthyroid cancer. Several NIS-based gene therapy experiments have found that NIS-mediated radioiodide uptake destroys malignant tumor cells (41, 42). In contrast, we reported that Na131I administration did not decrease the volumes of experimental tumors formed by malignantly transformed rat thyroid cells that were stably transfected with rat NIS cDNA (8). The effect of 131I is proportional to the effective radiation dose delivered to the tumor tissue, which depends on the effective half-life of 131I in the tumor’s 131I-concentrating ability (34). Furthermore, Schlumberger et al. (5) reported that the presence of 131I uptake that could be detected using whole body scanning was an important prognostic factor in thyroid cancer patients with metastasis. In the present study whole body scanning performed 48 h after radioiodide injection showed that AdTTF-1-treated tumors clearly demonstrated radioiodide accumulation, whereas AdLacZ-treated tumors did not. In addition, AdTTF-1 induced 7 times the protein-bound radioiodide accumulation as that induced by AdLacZ in xenotransplanted N-BHP18–21v cells. These results indicate that AdTTF-1 induced efficient iodide retention and organification. We hope that these findings will improve the efficacy of radioiodide therapy for radioiodide-resistant thyroid cancer.

In the present study no detectable induction of Tg or TPO gene expression was observed in ARO anaplastic thyroid cancer cells (TTF-1+, Pax-8–). It is possible that infection with AdTTF-1 has no effect on Pax-8-negative anaplastic carcinoma. Combination with HDACIs, which induce Tg, TPO, and NIS mRNA and protein expression in ARO anaplastic thyroid cancer cells (21), may confer beneficial effects on treatment with AdTTF-1.


   Footnotes
 
Abbreviations: AdTTF-1, Adenovirus vector containing thyroid transcription factor-1; CMV, cytomegalovirus; FCS, fetal calf serum; HBSS, Hanks’ balanced salt solution; HDACI, histone deacetylase inhibitor; MMI, methimazole; MOI, multiplicity of infection; NIS, sodium/iodide symporter; TCA, trichloroacetic acid; Tg, thyroglobulin; TPO, thyroperoxidase; TTF-1, thyroid transcription factor-1.

Received May 17, 2004.

Accepted for publication July 15, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. DeGroot LJ, Kaplan EL, McCormick M, Straus FH 1990 Natural history, treatment, and course of papillary thyroid carcinoma. J Clin Endocrinol Metab 71:414–424[Abstract]
  2. Beierwaltes WH 1978 The treatment of thyroid carcinoma with radioactive iodine. Semin Nucl Med 8:79–94[CrossRef][Medline]
  3. Schlumberger M, Tubiana M, De Vathaire F, Hill C, Gardet P, Travagli JP, Fragu P, Lumbroso J, Caillou B, Parmentier C 1986 Long-term results of treatment of 283 patients with lung and bone metastases from differentiated thyroid carcinoma. J Clin Endocrinol Metab 63:960–967[Abstract]
  4. Schlumberger MJ 1998 Papillary and follicular thyroid carcinoma. N Engl J Med 338:297–306[Free Full Text]
  5. Schlumberger M, Challeton C, De Vathaire F, Travagli JP, Gardet P, Lumbroso JD, Francese C, Fontaine F, Ricard M, Parmentier C 1996 Radioactive iodine treatment and external radiotherapy for lung and bone metastases from thyroid carcinoma. J Nucl Med 37:598–605[Abstract/Free Full Text]
  6. Nel CJ, van Heerden JA, Goellner JR, Gharib H, McConahey WM, Taylor WF, Grant CS 1985 Anaplastic carcinoma of the thyroid: a clinicopathologic study of 82 cases. Mayo Clin Proc 60:51–58[Medline]
  7. Venkataraman GM, Yatin M, Marcinek R, Ain KB 1999 Restoration of iodide uptake in dedifferentiated thyroid carcinoma: relationship to human Na+/I– symporter gene methylation status. J Clin Endocrinol Metab 84:2449–2457[Abstract/Free Full Text]
  8. Shimura H, Haraguchi K, Miyazaki A, Endo T, Onaya T 1997 Iodide uptake and experimental 131I therapy in transplanted undifferentiated thyroid cancer cells expressing the Na+/I– symporter gene. Endocrinology 138:4493–4496[Abstract/Free Full Text]
  9. Huang M, Batra RK, Kogai T, Lin YQ, Hershman JM, Lichtenstein A, Sharma S, Zhu LX, Brent GA, Dubinett SM 2001 Ectopic expression of the thyroperoxidase gene augments radioiodide uptake and retention mediated by the sodium iodide symporter in non-small cell lung cancer. Cancer Gene Ther 8:612–618[CrossRef][Medline]
  10. Mitchell PJ, Tjian R 1989 Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245:371–378[Abstract/Free Full Text]
  11. Damante G, Di Lauro R 1994 Thyroid-specific gene expression. Biochim Biophys Acta 1218:255–266[Medline]
  12. Guazzi S, Price M, De Felice M, Damante G, Mattei MG, Di Lauro R 1990 Thyroid nuclear factor 1 (TTF-1) contains a homeodomain and displays a novel DNA binding specificity. EMBO J 9:3631–3639[Medline]
  13. Zannini M, Avantaggiato V, Biffali E, Arnone MI, Sato K, Pischetola M, Taylor BA, Phillips SJ, Simeone A, Di Lauro R 1997 TTF-2, a new forkhead protein, shows a temporal expression in the developing thyroid which is consistent with a role in controlling the onset of differentiation. EMBO J 16:3185–3197[CrossRef][Medline]
  14. Zannini M, Francis-Lang H, Plachov D, Di Lauro R 1992 Pax-8, a paired domain-containing protein, binds to a sequence overlapping the recognition site of a homeodomain and activates transcription from two thyroid-specific promoters. Mol Cell Biol 12:4230–4241[Abstract/Free Full Text]
  15. Shimura H, Okajima F, Ikuyama S, Shimura Y, Kimura S, Saji M, Kohn LD 1994 Thyroid-specific expression and cyclic adenosine 3',5'-monophosphate autoregulation of the thyrotropin receptor gene involves thyroid transcription factor-1. Mol Endocrinol 8:1049–1069[Abstract]
  16. Kimura S 1997 Role of the thyroid-specific enhancer-binding protein in transcription, development and differentiation. Eur J Endocrinol 136:128–136[Medline]
  17. Endo T, Kaneshige M, Nakazato M, Ohmori M, Harii N, Onaya T 1997 Thyroid transcription factor-1 activates the promoter activity of rat thyroid Na+/I– symporter gene. Mol Endocrinol 11:1747–1755[Abstract/Free Full Text]
  18. Ros P, Rossi DL, Acebron A, Santisteban P 1999 Thyroid-specific gene expression in the multi-step process of thyroid carcinogenesis. Biochimie 81:389–396[Medline]
  19. Shimura H, Suzuki H, Miyazaki A, Furuya F, Ohta K, Haraguchi K, Endo T, Onaya T 2001 Transcriptional activation of the thyroglobulin promoter directing suicide gene expression by thyroid transcription factor-1 in thyroid cancer cells. Cancer Res 61:3640–3646[Abstract/Free Full Text]
  20. Ohta K, Pang XP, Berg L, Hershman JM 1997 Growth inhibition of new human thyroid carcinoma cell lines by activation of adenylate cyclase through the ß-adrenergic receptor. J Clin Endocrinol Metab 82:2633–2638[Abstract/Free Full Text]
  21. Furuya F, Shimura H, Suzuki H, Taki K, Ohta K, Haraguchi K, Onaya T, Endo T, Kobayashi T 2004 Histone deacetylase inhibitors restore radioiodide uptake and retention in poorly differentiated and anaplastic thyroid cancer cells by expression of the sodium/iodide symporter thyroperoxidase and thyroglobulin. Endocrinology 145:2865–2875[Abstract/Free Full Text]
  22. Van Herle AJ, Agatep ML, Padua III DN, Totanes TL, DVC, Van Herle HM, Juillard GJ 1990 Effect of 13 cis-retinoic acid on growth and differentiation of human follicular carcinoma cells (UCLA R0 82 W-1) in vitro. J Clin Endocrinol Metab 71:755–763[Abstract]
  23. Saito T, Endo T, Kawaguchi A, Ikeda M, Nakazato M, Kogai T, Onaya T 1997 Increased expression of the Na+/I– symporter in cultured human thyroid cells exposed to thyrotropin and in Graves’ thyroid tissue. J Clin Endocrinol Metab 82:3331–3336[Abstract/Free Full Text]
  24. Saito T, Endo T, Kawaguchi A, Ikeda M, Katoh R, Kawaoi A, Muramatsu A, Onaya T 1998 Increased expression of the sodium/iodide symporter in papillary thyroid carcinomas. J Clin Invest 101:1296–1300[Medline]
  25. Graham FL, van der Eb AJ 1973 Transformation of rat cells by DNA of human adenovirus 5. Virology 54:536–539[CrossRef][Medline]
  26. Sambrook J, Russell DW 2001 Molecular cloning: a laboratory manual. 3rd ed. Plainview, NY: Cold Spring Harbor Laboratory Press
  27. Ohta K, Endo T, Onaya T 1991 The mRNA levels of thyrotropin receptor, thyroglobulin and thyroid peroxidase in neoplastic human thyroid tissues. Biochem Biophys Res Commun 174:1148–1153[CrossRef][Medline]
  28. Miyazaki A, Shimura H, Endo T, Haraguchi K, Onaya T 1999 Tumor necrosis factor-{alpha} and interferon-{gamma} suppress both gene expression and deoxyribonucleic acid-binding of TTF-2 in FRTL-5 cells. Endocrinology 140:4214–4220[Abstract/Free Full Text]
  29. Weiss SJ, Philp NJ, Grollman EF 1984 Iodide transport in a continuous line of cultured cells from rat thyroid. Endocrinology 114:1090–1098[Abstract]
  30. Fagin JA, Tang SH, Zeki K, Di Lauro R, Fusco A, Gonsky R 1996 Reexpression of thyroid peroxidase in a derivative of an undifferentiated thyroid carcinoma cell line by introduction of wild-type p53. Cancer Res 56:765–771[Abstract/Free Full Text]
  31. Schmutzler C 2001 Regulation of the sodium/iodide symporter by retinoids: a review. Exp Clin Endocrinol Diabetes 109:41–44[CrossRef][Medline]
  32. Miccadei S, De Leo R, Zammarchi E, Natali PG, Civitareale D 2002 The synergistic activity of thyroid transcription factor 1 and Pax 8 relies on the promoter/enhancer interplay. Mol Endocrinol 16:837–846[Abstract/Free Full Text]
  33. 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]
  34. 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]
  35. Yoshida A, Sasaki N, Mori A, Taniguchi S, Ueta Y, Hattori K, Tanaka Y, Igawa O, Tsuboi M, Sugawa H, Sato R, Hisatome I, Shigemasa C, Grollman EF, Kosugi S 1998 Differences in the electrophysiological response to I– and the inhibitory anions SCN- and ClO-4, studied in FRTL-5 cells. Biochim Biophys Acta 1414:231–237[Medline]
  36. Rodriguez AM, Perron B, Lacroix L, Caillou B, Leblanc G, Schlumberger M, Bidart JM, Pourcher T 2002 Identification and characterization of a putative human iodide transporter located at the apical membrane of thyrocytes. J Clin Endocrinol Metab 87:3500–3503[Abstract/Free Full Text]
  37. Lamas L, Dorris ML, Taurog A 1972 Evidence for a catalytic role for thyroid peroxidase in the conversion of diiodotyrosine to thyroxine. Endocrinology 90:1417–1426[Medline]
  38. Degroot LJ, Niepomniszcze H 1977 Biosynthesis of thyroid hormone: basic and clinical aspects. Metabolism 26:665–718[CrossRef][Medline]
  39. Ekholm R, Bjorkman U 1984 Localization of iodine binding in the thyroid gland in vitro. Endocrinology 115:1558–1567[Abstract]
  40. 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]
  41. Mandell RB, Mandell LZ, Link Jr CJ 1999 Radioisotope concentrator gene therapy using the sodium/iodide symporter gene. Cancer Res 59:661–668[Abstract/Free Full Text]
  42. Cho JY, Xing S, Liu X, Buckwalter TL, Hwa L, Sferra TJ, Chiu IM, Jhiang SM 2000 Expression and activity of human Na+/I– symporter in human glioma cells by adenovirus-mediated gene delivery. Gene Ther 7:740–749[CrossRef][Medline]



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