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Endocrinology Vol. 142, No. 8 3369-3379
Copyright © 2001 by The Endocrine Society

ARTICLES

Differential Regulation of the Human Sodium/Iodide Symporter Gene Promoter in Papillary Thyroid Carcinoma Cell Lines and Normal Thyroid Cells

Takahiko Kogai, Jerome M. Hershman, Katsuaki Motomura, Toyoshi Endo, Toshimasa Onaya and Gregory A. Brent

The Endocrinology Division (T.K., J.M.H., K.M., G.A.B.), VA Greater Los Angeles Healthcare System and Department of Medicine, UCLA School of Medicine, Los Angeles, California 90073; and Third Department of Internal Medicine (T.E., T.O.), Yamanashi Medical University, Tamaho, Yamanashi 409-3898, Japan

Address all correspondence and requests for reprints to: Gregory A. Brent, M.D., Molecular Endocrinology Laboratory, VA Greater Los Angeles Healthcare System, 11301 Wilshire Boulevard, Building 114, Room 230, Los Angeles, California 90073. E-mail: gbrent{at}ucla.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The absence of TSH-stimulated radioiodide uptake in differentiated thyroid cancer is associated with a high recurrence rate and reduced survival. We studied regulation of the sodium/iodide symporter gene in human papillary thyroid cancer cell lines (BHP) and primary human thyroid cells. BHP cells expressed very low levels of sodium/iodide symporter mRNA and did not concentrate iodide, but iodide uptake was restored to levels seen in FRTL-5 rat thyroid cells by stable transfection of a sodium/iodide symporter cDNA. Sodium/iodide symporter gene expression, therefore, was necessary and sufficient for iodide uptake in BHP cells. We cloned the human sodium/iodide symporter gene 5'-flanking region and analyzed progressive 5'-deletions in transient transfections. We identified a region, -596 to -268, essential to confer full promoter activity in primary normal human thyroid cells. Sodium/iodide symporter promoter activity in four BHP cell lines, however, was markedly reduced, consistent with down-regulation of the endogenous sodium/iodide symporter gene. Nuclear extracts from BHP 2–7 cells had reduced or absent binding to regions of the sodium/iodide symporter promoter shown to be critical for expression, compared with nuclear extracts from FRTL-5 cells. Competition studies indicated that these nuclear proteins were not known thyroid transcription factors. Modifications of the sodium/iodide symporter promoter with demethylation or histone acetylation did not increase sodium/iodide symporter expression, and no deletions of the critical regulatory region were identified in the endogenous gene in BHP cells. Regulation of the sodium/iodide symporter 5'-flanking region in transient transfection paralleled endogenous sodium/iodide symporter expression. Reduced expression of potential novel nuclear factor(s) in these cell lines may contribute to reduced sodium/iodide symporter expression resulting in absence of iodide uptake in some papillary thyroid cancers.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RADIOIODIDE THERAPY HAS been used for over 40 yr in patients with differentiated thyroid carcinoma (1). Radioactive iodine is transported into cancer cells via the sodium/iodide symporter on the plasma membrane and exerts a local destructive effect, delivering radiation doses of 20,000 rads (200 grays) or more (2). However, many metastatic thyroid carcinomas do not concentrate sufficient 131I for therapy (3). In addition, some tumors that initially concentrate radioiodine lose differentiated function and are unable to concentrate iodine. Patients with 131I-resistant differentiated carcinomas have a high recurrence rate and reduced survival, although the mechanism responsible for deficient iodine accumulation in such carcinomas is not well known.

Reduced expression of sodium/iodide symporter (NIS) mRNA has been reported in some thyroid carcinoma specimens compared with normal thyroid tissue (4, 5, 6, 7). Using RT-PCR, NIS mRNA was undetectable in 17–33% of papillary cancer specimens (4, 5, 6). In one study, NIS mRNA was not detected in three follicular carcinomas (5). Therefore, reduced iodide transport in some thyroid cancer may be due to reduced NIS gene expression.

Regulatory regions in rat NIS promoter have been characterized (8, 9, 10, 11). The cAMP response element is not found in the minimal region required for expression (8, 9), although rat NIS expression is markedly stimulated by TSH (12, 13), forskolin, and (Bu)2cAMP (12). Thyroid transcription factor-1 (TTF-1), a homeo-box containing protein, binds to the sequence between -245 and -230 in a cell-specific manner and stimulates NIS promoter activity (9). The thyroid specific enhancer region is located between -2495 and -2264 of the rat NIS gene 5'-flanking region, and Pax-8, a paired-domain transcription factor, binds to the upstream enhancer (11). TSH/cAMP-induced up-regulation of the rat NIS gene expression requires a novel thyroid transcription factor, NIS TSH-responsive factor-1 (NTF-1), which also appears to be involved in TTF-1-mediated thyroid-specific NIS gene expression (10). The human NIS gene promoter has been reported to contain putative TTF-1 binding and Pax-8 binding sites (14, 15, 16).

We have established four human papillary thyroid cancer cell lines, BHP 2–7, 7–13, 10–3, and 18–21 (17). All of the cell lines express Pax-8 mRNA (17). BHP cells cannot accumulate radioiodine, and NIS mRNA has not been detected by Northern blot analysis (17). These observations suggest that the absence of iodide transport in these thyroid cancer cell lines is due to reduced NIS expression. Reduced NIS expression may be the result of modification of the promoter, inadequate or defective endogenous TTF-1 or Pax-8, absence of a novel transcription factor, or overexpression of a repressor. In the present study, we tested these possibilities with an independently cloned human NIS promoter and compared expression in long-term cultured normal human thyroid cells (18, 19, 20, 21) and the FRTL-5 rat thyroid cell line (22) to the BHP papillary thyroid cancer cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
BHP 2–7, 7–13, 10–3, and 18–21 cell lines, derived from human papillary thyroid cancer (17), were grown in Roswell Park Memorial Institute 1640 medium (Sigma, St. Louis, MO) supplemented with 8% FCS (Life Technologies, Inc., Grand Island, NY). FRTL-5 rat thyroid cells, kindly provided by Dr. L. D. Kohn (NIH, Bethesda, MD), were maintained with 1 mU/ml bovine TSH, as previously described (22). Human primary thyroid cells were prepared and maintained as previously described (18, 21). Briefly, thyroid tissue, obtained from the normal, nonaffected lobe in eight patients with papillary carcinoma or follicular adenoma who underwent thyroidectomy, was freed from adherent connective tissue, cut in small (less than 1 mm diameter) pieces, and washed in Ca2+- and Mg2+-free HBSS. The enzymatic digestion was performed for 2 h with a solution consisting of 20 U/ml collagenase (Sigma), 0.75 mg/ml trypsin (Life Technologies, Inc., 1:300), and 2% heat-inactivated dialyzed chicken serum (Life Technologies, Inc.) in Ca2+- and Mg2+-free HBSS (CTC). Cells were seeded at a density of 105 per 100-mm dish. Released cells were grown in Coon’s modified Ham’s F-12 medium (Sigma) supplemented with 5% FCS (Life Technologies, Inc.), bovine insulin (1 µg/ml) (Sigma), bovine transferrin (5 µg/ml) (Sigma), and hydrocortisone (0.01 nM) (Sigma). Freshly frozen bovine hypothalamus and bovine pituitary (Pel Freeze Biologicals, Rogers, AR) extracts were prepared as previously described (18), and added to final concentration of 75 and 5 µg of protein per milliliter of the medium, respectively. Before utilization of the primary cells, thyroglobulin production was confirmed with a commercial kit (Diagnostic Products Corp., Los Angeles, CA), according to the manufacturer’s instructions, as described (18). Use of human surgical material was approved by the institutional human subjects protection committee.

Follicle induction
Follicle induction was carried out as described (21). Briefly, monolayer cells cultured for 1–2 months were digested with CTC and plated at a density of 106 per 100 mm agarose-coated dish and maintained in the follicle-induction medium, which consists of Coon’s modified Ham’s F-12 medium supplemented with 0–1% FCS, bovine insulin (1 µg/ml), bovine transferrin (5 µg/ml), hydrocortisone (0.01 nM) with 0.1 mU/ml bovine TSH (Sigma). After the seeding, the long term-cultured normal human thyroid cells associated into globular aggregates in 18–24 h, and periodic acid schiff staining and electron microscopy demonstrated follicle formation (21).

Iodide uptake assay
To study thyroid follicles, cells in agarose-coated dishes were transferred to 1.5 ml microtubes, centrifuged and washed with HBSS. The cells were then incubated with 100 nmol/liter 125I- (50 mCi/mmol) (ICN Biomedicals, Costa Mesa, CA) in HBSS at 37 C on agarose-coated 12-well plates. After 2 h incubation, the cells were collected in 1.5 ml microtubes, centrifuged, and rapidly rinsed with HBSS twice. The radioactivity of the pellet was measured with a {gamma}-counter, and normalized to the cellular protein content measured in the same cells with protein assay (Bio-Rad Laboratories, Inc., Richmond, CA) protein assay. For monolayer cells, the assay was performed with 100 nmol/liter 125I- (50 mCi/mmol) as described previously (23, 24).

Stable expression of human NIS in BHP cells
Human NIS cDNA in pcDNA3 (24), mammalian expression vector with cytomegalovirus (CMV) promoter and Neomycin resistant gene, was transfected into BHP 2–7, 7–13, and 18–21 cells by Gene Pulser (Bio-Rad Laboratories, Inc.) at 280 V and 960 microfarads. Four weeks after transfection, 5 G-418-resistant clones were pooled and used for iodide uptake assays as described previously (24, 25). As a control, the pcDNA3 empty vector was also transfected into these cells.

Northern blot analysis
Total RNA (20 µg) was prepared with Trizol reagent (Life Technologies, Inc.) from cultured cells, separated on a 1% agarose gel containing formaldehyde, and transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH) as described previously (26). A human NIS cRNA probe was prepared by the in vitro transcription of pcDNA3 containing the human NIS cDNA with SP6 RNA polymerase (Promega Corp., Madison, WI) as described previously (24, 27). The filters were hybridized with the probe. Intensities of 28s ribosomal RNA in the agarose gels and NIS mRNA signals on the blots were quantitated on a Macintosh computer from scanned images using the NIH Image program version 1.61 (developed at the U.S. NIH and available on the Internet at http://rsb.info.nih.gov/nih-image/).

Isolation of the human NIS gene
A {lambda} FixII human lung fibroblast genomic library was screened for the NIS gene using 32P-labeled rat NIS cDNA that contains full coding sequence (24). One of the positive clones (clone {lambda} II-1) was subcloned into pBluescript SK+ (Stratagene, La Jolla, CA) by NotI. Selected restriction fragments were further subcloned into pBluescript SK+ or M13 phage and sequenced by the dideoxynucleotide method.

Reporter plasmids and cDNA expression vectors
DNA fragments from the human NIS 5'-flanking region [nucleotides (nt) -1622 to -268, nt -812 to -268, and nt -596 to -268; the A in the ATG initiation codon is designated as +1] were amplified from the pBluescript-{lambda} II-1 clone by PCR with forward primers containing BglII site (5'-GTCAAGATCTTACAGGCATGTGCCACC for -1622 fragment, 5'-TGATCAGATCTTTGGGGGTGGTAAAGCCAG for -812 fragment, and 5'-TGATCAGATCTGTGATCAGGGGATCACA for -596 fragment) and reverse primer containing HindIII site (5'-GATCAAGCTTCTCACTCTGGGTTC). They were inserted into the BglII- HindIII site of pGL3-Basic vector (Promega Corp.) containing the luciferase reporter gene. These luciferase constructs were designated p{Delta}1622-Luc, p{Delta}812-Luc, and p{Delta}596-Luc. SacI site (-729) and SmaI site (-415) are on the human NIS promoter. p{Delta}812-Luc was cleaved by SacI or SmaI and fragments that contain sequence from -729 to -268 or from -415 to -268 were self-ligated. These constructs were designated p{Delta}729-Luc and p{Delta}415-Luc. All the sequence of the fragment generated by PCR was confirmed by sequencing after cloning.

A 5'-upstream sequence of the NIS gene from -2829 to -1578 was amplified from genomic DNA of MCF-7 human breast cancer cells, which express functional NIS (28), using the following oligonucleotides: 5'-GATCGGTACCGCATATGTTTACATTGGTCGGCAGG, 5'-GATCGCTAGCTGAGTCCCAGAATTCGAGACTACC. The amplified DNA fragment was cloned upstream of the SV40 promoter in pGL3 Promoter vector (Promega Corp.) using MluI and NheI.

A human Pax-8 expression vector, pSVK3PAX8 (29), was provided by Dr. G. F. Saunders (The University of Texas M.D. Anderson Cancer Center, Houston, TX). A rat TTF-1 expression vector, pRcCMV-THA (30), was provided by Dr. R. Di Lauro (Naples, Italy). A luciferase reporter gene construct with 6.3 kb of upstream sequence of the human thyroperoxidase (TPO) gene, pSVOAL-A{Delta}5'-TPO (31), was provided by Dr. S. Kimura (National Cancer Institute, Bethesda, MD). Human NIS cDNA in pcDNA3 (Invitrogen, Carlsbad, CA), a human NIS expression vector, was prepared as previously described (24).

Transient transfection expression analysis
BHP cells (1.5 x 105), FRTL-5 cells (2 x 105) and human primary normal thyroid cells (2 x 105) were seeded in 35 mm-diameter dishes before transfection (24 h, 48 h, and 48 h, respectively). Unless otherwise noted, 0.5 µg luciferase construct and 0.5 µg pSV-ß-Galactosidase Control Vector (Promega Corp.) were transfected into BHP or FRTL-5 cells by LipofectAMINE Plus reagent (Life Technologies, Inc.), and luciferase assay was performed 48 h after the beginning of transfection with commercial reporter lysis buffer and the luciferase assay substrate (both from Promega Corp.). For primary thyroid cells, 1 µg luciferase construct and 1 µg pSV-ß-Galactosidase Control Vector were transfected by LipofectAMINE reagent, and luciferase assay was performed 48 h after the beginning of transfection as described above. ß-Galactosidase assay was performed as described previously (10), and the transfection efficiency of luciferase reporter constructs was normalized to ß-galactosidase activity.

Stable transfection expression analysis
To establish BHP cells stably transfected with a luciferase gene under the control of human NIS promoter, 40 µg of the p{Delta}812-Luc construct was transfected into BHP 2–7 cells with 5 µg of pcDNA3 (empty) vector by electroporation, as described above. Four weeks after the transfection, 48 clones selected by G-418 (Life Technologies, Inc.) were pooled and luciferase activity was evaluated. Six positive clones were used for transient expression studies with vectors expressing TTF-1 or Pax-8.

Genomic PCR and sequencing
Genomic DNA was extracted from BHP cells by proteinase K digestion and phenol/chloroform extraction as described previously (32). Genomic DNA (2 µg) was digested by 40 U of HindIII, and the DNA fragment from -812 to -268 was amplified by PCR with the same primers used for construction of the reporter vector. The fragment was used for direct cycle sequencing with the same primers.

Nuclear extract preparation
BHP cells, grown to 80% confluence, were washed with PBS, pH 7.4, collected by scraper and centrifugation, and suspended in five pellet volumes of 0.3 M sucrose and 2% Tween-40 in buffer A [10 mM HEPES-KOH, pH 7.9, containing 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonylfluoride, 2 µg/ml leupeptin, and 2 µg/ml pepstatin-A]. After the cells were frozen in liquid nitrogen, thawed, and gently homogenized, the suspension was layered onto 1.5 M sucrose in buffer A and centrifuged at 32,000 x g in a swinging bucket rotor. Nuclei were washed with buffer A and lysed in 2.5 vol. of buffer B [10 mM HEPES-KOH, pH 7.9, containing 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0.5 mM DTT, 0.5 mM phenylmethylsulfonylfluoride, 2 µg/ml leupeptin, and 2 µg/ml pepstatin-A]. Lysed nuclei were centrifuged at 15,000x g for 1 h and used in EMSAs. Preparation of nuclear extract from FRTL-5 cells was the same as that for BHP cells except that the centrifugation for nuclei purification was at 25,000 x g.

EMSA
EMSAs were performed as previously described (9, 10). Briefly, synthesized double-stranded oligonucleotides were labeled with {gamma}-32P ATP (ICN Biomedicals) by T4 polynucleotide kinase, and purified on a 6% native polyacrylamide gel. Nuclear extract (2 µg) was incubated in a 15-µl reaction volume for 20 min at room temperature in the following buffer with or without unlabeled competitor oligonucleotides: 10 mM Tris-HCl (pH 7.6), 50 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 5% glycerol, 0.1% Triton X-100, and 2 µg polydeoxyinosinic-deoxycytidylic acid. Labeled probe (50, 000 cpm; ~0.5 ng DNA) was added, and incubation was continued for an additional 20 min at room temperature. DNA-protein complexes were separated on 5% native polyacrylamide gels.

To demonstrate binding of TTF-1 or Pax-8 protein, the oligo C from the rat thyroglobulin promoter, containing an overlapping TTF-1 and Pax-8 element (33), or the PA oligonucleotide from the rat NIS promoter enhancer, containing a Pax-8 element (11), was used. Supershift assays were carried out as previously described (34) with anti-TTF-1 (35) and Pax-8 (34) antibodies.

Statistical analysis
Statistical significance was determined by a paired t test using STAT VIEW software (Abacus Concept, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Iodide uptake by BHP cells and long-term cultured normal human thyroid cells
We compared iodide uptake in human papillary thyroid cancer-derived BHP cells with that in long term-cultured normal human thyroid cells (18). Normal human thyroid cells, but not BHP cell lines, concentrated iodide (Fig. 1AGo), consistent with previous reports (17, 21). We recently found that long-term cultured normal human thyroid cells can accumulate significantly more iodide when grown as follicles than as monolayers, without a change in NIS mRNA or protein levels (21). To test whether induction of three dimensional structure in cultured BHP cells stimulates iodide uptake, BHP cells were seeded into agarose-coated dishes and incubated with follicle-induction medium as described (21), and iodide uptake assays were performed. All BHP cell lines formed three dimensional branching structures (Fig. 1CGo); however, the iodide accumulation remained very low (about 5% to 8% of uptake in normal human thyroid cells) (Fig. 1AGo).



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  		Figure 1. A, Iodide uptake in normal human primary thyroid cells (normal) and four BHP cell lines (2 3 4 5 6 7 7 8 9 10 11 12 13 18 19 20 21 ) in monolayer and follicle-forming conditions. Monolayers (white bar); after long-term culture (34–56 days) in the growth medium, the medium was switched to the follicle-induction medium (serum-free) with 0.1 mU/ml TSH in the monolayer condition for 72 h, and the iodide uptake assay was performed. Follicles (black bar); after long-term culture (34–56 days) in the growth medium, cells were induced to form follicles on agarose-coated dishes with the follicle-induction medium. They were incubated with 0.1 mU/ml TSH for 72 h and iodide uptake was measured. The radioactivity was normalized to the cellular protein content measured in the same cells. Values are the mean ± SE (n = 4). * and **, Significantly higher (P < 0.05 and P < 0.0001, respectively) compared with BHP cells in the same condition. B and C, Photomicrograph of the BHP 2–7 cells in the monolayer (B) and follicle-forming (C) conditions (x200 magnification).

 
NIS mRNA levels in BHP cell lines
We next compared NIS mRNA levels in BHP cells with that in normal human thyroid cells, maintained as monolayers. We used a human NIS cRNA probe and detected a single band of NIS mRNA in BHP cells (Fig. 2Go), although the level of NIS mRNA in BHP cells was only 4–13% of that in normal human thyroid cells. In the previous report, NIS mRNA was not detected in BHP cells by Northern blot analysis with a rat NIS cDNA probe (17).



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  		Figure 2. NIS mRNA expression in long-term cultured normal human thyroid cells (normal) and four BHP cell lines (2 3 4 5 6 7 7 8 9 10 11 12 13 18 19 20 21 ). Upper panel, Hybridization with a 32P-labeled human NIS RNA probe. The experiment is representative of three experiments performed with similar results. The lower panel displays an image of the respective ethidium bromide-stained nitrocellulose membrane used for hybridization.

 
Stable expression of NIS restores iodide accumulation in BHP cells
To investigate whether NIS expression could restore iodide accumulation in BHP cells, we stably transfected human NIS cDNA as described previously (24). Iodide transport was fully restored in BHP 2–7 cells (Fig. 3Go) and in other BHP cell lines (7, 8, 9, 10, 11, 12, 13) (data not shown). These results indicate that the expression of NIS is necessary and sufficient for iodide transport in these cells.



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  		Figure 3. Uptake of iodide by BHP 2–7 cells that stably expressed recombinant human NIS, compared with FRTL-5 rat thyroid cells. BHP 2–7 cells were transfected with (•) or without ({square}) human NIS cDNA in pcDNA3. FRTL-5 (x) cells were maintained with 1.0 mU/ml TSH. Cells grown in 12-well dishes were incubated with 100 nmol/liter 125I- (50 mCi/mmol) (ICN Biomedicals) in HBSS at 37 C for the indicated times, and then the content of iodide in the cells was determined with {gamma}-counter. Nonspecific binding of 125I- was determined in duplicate assays in the presence of 30 µM KClO4, and subtracted from the values measured (23 ). Values are the mean ± SE (n = 3).

 
Isolation and sequencing of a genomic fragment containing the 5'-flanking region of the human NIS gene
To study the regulation of NIS gene expression in the BHP cell lines and normal thyroid cells, we cloned the 5'-flanking region of the human NIS gene. Two positive clones were isolated by screening a human lung fibroblast genomic library (1.5 x 106 plaques) with 32P-labeled rat NIS cDNA (-29 to +1975 bp; the A in the ATG initiation codon is designated as +1). Sequencing of the XbaI-KpnI fragment showed that it contains the translation initiation site and that the clone {lambda} II -1 contains about 2.2 kb upstream region of the human NIS promoter. The sequence from the XbaI site (-847 nt) to the ATG was the same as that previously reported (16), but three mismatches were found compared with the data of Ryu et al. (15); -648 (C to T), -646 (C to T), and -567 nt (G to T). The same sequence was shown by the direct sequencing of the gene from the BHP cells as described below.

Identification of cell type-specific promoter activity in the 5'-flanking region of human NIS gene
To investigate the location of enhancer element(s) in the upstream regulatory sequence and required for NIS gene expression, we made a series of luciferase reporter constructs containing progressive 5'-deletions of the human NIS gene 5'-flanking region (Fig. 4AGo). These constructs were tested in the BHP cell lines and normal human thyroid cells.



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  		Figure 4. Human NIS promoter activity in long-term cultured normal human thyroid cells and four BHP cell lines. A, Schematic representation of the location of putative transcription factor binding sites and expression vector constructs used in transfection experiments. The 5'-flanking region of the human NIS gene and various deletions were ligated to the luciferase gene in pGL3-Basic vector. The numbering of nucleotides refers to their positions relative to the translation start codon of the NIS gene. Putative transcription factor binding sites are indicated by a thick line for Pax-8, hatched line for TTF-1, and broken line for NTF-1. The putative transcription initiation sites are indicated by arrows [-375, Ryu et al. (15 ); -336, Venkataraman et al. (16 )]. B, Promoter activity of the human NIS promoter-luciferase chimeric plasmids in normal human follicular cells and BHP cells. The NIS promoter activity is reduced in all BHP cell lines. The graph shows the relative luciferase activity in normal human thyroid follicular cells or 4 BHP cell lines after transfection with the NIS-luciferase deletion mutants as indicated. All cells were cotransfected with the pSV-ß-Galactosidase Control vector, and the transfection efficiency was normalized to ß-Galactosidase activity. *, Significant difference (P < 0.01) compared with BHP cells in the same condition.

 
The -812, -729, and -596 constructs expressed luciferase activity significantly greater than basal levels (pGL3-Basic empty vector) in normal human thyroid cells (P < 0.0001), whereas p{Delta}415-Luc did not express above background (Fig. 4BGo). These results indicate that the sequence in the region -596 to -268 is essential to confer full promoter activity. Deletion of the sequence -596 to -415 reduced expression of the reporter to that of the promoterless vector. NIS 5'-deletion reporter constructs transfected in the BHP cell lines expressed significantly less (12–40%; P < 0.01) compared with that in normal thyroid cells. The activity of the promoter closely paralleled endogenous NIS gene expression in normal and thyroid cancer cells.

The 5'-flanking region between -2,829 and -268 was studied in two segments and transfected in FRTL-5 and BHP 2–7 cells. The 5'-flanking region -1,622 to -268 (containing the -596 to -415 enhancer region and basal NIS promoter) was inserted into a luciferase reporter construct and expressed at a higher level in FRTL-5 cells compared with BHP 2–7 cells (data not shown). The region -2,829 to 1,578 was ligated upstream of the SV40 promoter and did not show significantly different expression between the cell lines (data not shown).

Effects of overexpression of TTF-1 or Pax-8 on human NIS promoter activity
Although the rat NIS promoter can be stimulated by TTF-1 (9), only weak elements for TTF-1 are found by sequence inspection in the human promoter between -729 and -415 (see Fig. 4AGo). There are, however, two putative elements for Pax-8 in this region (15, 16). We first characterized the presence of endogenous Pax-8 and TTF-1 protein in BHP 2–7 and FRTL-5 cells. Nuclear extract from both cell types were bound to a rat thyroglobulin promoter oligonucleotide, containing a TTF-1/Pax8 element (33), and EMSA was performed with antibody supershift (Fig. 5Go). FRTL-5 cells contained both TTF-1 and Pax-8 protein, and BHP 2–7 cells only Pax-8 protein.



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  		Figure 5. Endogenous expression of Pax-8 and TTF-1 protein in BHP 2–7 cells and FRTL-5 cells. Radiolabeled rat thyroglobulin promoter oligonucleotide, containing TTF-1 and Pax-8 elements (33 ), was incubated with nuclear extracts from FRTL-5 cells (lanes 1, 2, and 3) or BHP 2–7 cells (lane 4 and 5) in the presence (+) or absence (-) of anti-Pax-8 (lane 3 and 5) or TTF-1 (lane 2) antibodies. EMSA was performed as described in Materials and Methods.

 
Because TTF-1 protein levels might be limiting for human NIS promoter activation in BHP cells, we performed cotransfection studies with TTF-1 and Pax-8 expressing vectors. Although Pax-8 protein was detected in BHP cells, the endogenous protein may have reduced function. We first tested the human TPO promoter in BHP cells to confirm functional protein from our Pax-8 and TTF-1 expression vectors and to determine whether BHP cells contained any inhibitors to their action. The TPO promoter has been reported to be enhanced by TTF-1 (31) and Pax-8 (36) in nonthyroidal cells. Cotransfection of pSVK3PAX8 (29), human Pax-8 expression vector, or pRcCMV-THA (30), rat TTF-1 expression vector, with the human TPO promoter significantly (6.2 and 1.9-fold, respectively) increased the promoter activity in BHP 2–7 cells (Fig. 6Go, A and B). We next studied the NIS promoter in BHP cells. Cotransfection of pSVK3PAX8 or pRcCMV-THA with p{Delta}729-Luc did not significantly increase luciferase activity in BHP 2–7 or 18–21 cells (Fig. 7Go, A and B). We additionally studied BHP 2–7 cells stably transfected with the p{Delta}812-Luc reporter construct. Luciferase activity was not increased by transient expression of TTF-1 or Pax-8 (data not shown).



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  		Figure 6. Effects of cotransfection of TTF-1 or Pax-8 on human TPO promoter expression. A vector containing 6.3 kb of upstream sequence of the human TPO gene connected to a luciferase reporter gene (1 µg) was cotransfected with indicated amount of pRc/CMV-THA, a rat TTF-1 expression vector (A), or pSVK3PAX8, a human Pax-8 expression vector (B) into BHP 2–7 cells. Empty vectors (pRc/CMV or pSVK3 vector) were used to adjust the total amount of DNA transfected. Luciferase assay was performed 48 h after the transfection. All cells were cotransfected with the pSV-ß-Galactosidase Control vector, and the transfection efficiency was normalized to ß-Galactosidase activity. Values are the mean ± SE (n = 3). *, **, and ***, Significant difference (P < 0.02, P < 0.03, and P < 0.05, respectively) compared with BHP cells transfected with only the TPO promoter construct.

 


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  		Figure 7. Effects of cotransfection of TTF-1 or Pax-8 on human NIS promoter expression. The luciferase-NIS construct, p{Delta}729-Luc, was cotransfected with indicated amount of pRc/CMV-THA (A) or pSVK3PAX8 (B) into primary human thyroid cells, BHP 2–7 cells, or BHP 18–21 cells. Empty vectors (pRc/CMV or pSVK3 vector) were used to adjust the total amount of DNA transfected. Luciferase assay was performed 48 h after the transfection. All cells were cotransfected with the pSV-ß-Galactosidase Control vector, and the transfection efficiency was normalized to ß-Galactosidase activity. Values are the mean ± SE (n = 3).

 
Identification of nuclear factor(s) required for human NIS expression but the expression of which is reduced in BHP cells
To investigate whether the reduced NIS promoter expression in BHP cells is associated with altered binding of nuclear proteins, we made four contiguous oligonucleotides spanning the critical enhancer region -596 to -415; probe A, -596 to -531, which contains putative Pax-8 and NTF-1 [NIS-TSH responsive factor-1] (10) response elements; probe B, -530 to -471; probe C, -470 to -440, which contains a putative Pax-8 response element; probe D, -445 to -415, which contains a putative NTF-1 element and GC-box motif (see Fig. 8AGo) and compared the pattern of nuclear extract binding in EMSAs. Nuclear extracts from FRTL-5 cells, in which the human NIS promoter is active (data not shown) (14), formed two specific DNA-protein complexes with probe A (-596 to -531), which were not seen with nuclear extract from BHP 2–7 cells (Fig. 8BGo). The specific bands observed with probe A were not competed by 250-fold excess of the rat thyroglobulin promoter TTF-1/Pax-8 element (33), or a rat NTF-1 element (10) (Fig. 8CGo). Probe A contains putative Pax-8 and NTF-1 elements. No complexes were seen bound to probe B (-530 to -471) (data not shown). Nuclear extracts from both cell lines formed two specific complexes with probe C (-470 to -440); the intensity of the more slowly migrating band in FRTL-5 cells was about 2-fold greater than that seen with BHP 2–7 cell extract (Fig. 8DGo). The specific bands observed with probe C were not competed by 250-fold excess of oligonucleotides containing the Pax-8 element from the rat NIS promoter enhancer (11) (Fig. 8DGo). Nuclear extracts from both cell lines had the same binding pattern to probe D (-445 to -415) (data not shown). The two specific bands seen with probe D were not competed by 250-fold excess of the rat NTF-1 element. The ability of the rat NIS enhancer Pax-8 element to bind Pax-8 protein was confirmed by labeling the element and adding nuclear extract from BHP 2–7 and FRTL-5 cells. A complex was found that was supershifted with anti-Pax-8 antibody and competed with cold oligonucleotide (Fig. 9Go). The Pax-8 DNA complex band was more intense in BHP 2–7 cells compared with FRTL-5 cells. These observations demonstrated that nuclear factors which bind to probe A or probe C are absent (probe A) or reduced (probe C) in BHP 2–7 cells compared with FRTL-5 cells, and these factors are not TTF-1 or Pax-8. Furthermore, putative Pax-8 elements between -596 and -415 do not bind endogenous Pax-8 from BHP 2–7 cells.



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  		Figure 8. EMSAs with 4 contiguous oligonucleotides spanning the critical region of the human NIS promoter. Panel A, 4 oligonucleotides; probe A, -596 to -531, which contains a putative Pax-8 and NTF-1 response element; probe B, -530 to -471; probe C, -470 to -440, which contains a putative Pax-8 response element; probe D, -445 to -415, which contains a putative NTF-1 element and GC-box motif. Panels B–D, EMSAs with 32P-labeled oligonucleotides (probe A [panels B and C] and probe C [panel D]) and nuclear extracts from BHP 2–7 cells or FRTL-5 cells. Competitor fragments were the unlabeled oligonucleotides probe (self), the rat NTF-1 element (rat NTF-1), rat thyroglobulin promoter oligonucleotide containing TTF-1 and Pax-8 element (Tg-oligo C), or the rat NIS enhancer containing a Pax-8 element (Pax-8). The amount of cold oligonucleotide added as a competitor is indicated. The intensity of the bands {alpha} and ß in panel D was quantified by NIH image version 1.61 on a Macintosh computer (data not shown). A representative example of three experiments with similar results is shown.

 


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  		Figure 9. EMSAs with the rat NIS enhancer containing a Pax-8 element (11 ). 32P-labeled DNA fragment of rat NIS enhancer incubated with nuclear extracts from FRTL-5 or BHP 2–7 cells in the presence (+) or absence (-) of anti-Pax-8 antibody or self competitor (250-fold excess).

 
Deletion or mutation of human NIS promoter in BHP cells
To analyze the endogenous sequence in the human NIS gene regulatory region, we performed genomic PCR and direct sequencing. As shown in Fig. 10Go, the PCR analysis, using genomic DNA from BHP cells as templates, produced the same size fragments from -812 to -268 as the PCR products derived from the original human NIS gene clone, {lambda} II-1. This indicates that BHP cells have no significant deletion of the NIS gene promoter. We also analyzed for polymorphisms in this region using a direct sequencing technique. No mutation or deletion was found in the sequence between -730 and -349 in the BHP cells (data not shown).



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  		Figure 10. PCR analysis of endogenous NIS gene in BHP cell lines. Genomic DNA extracted from 4 BHP cell lines were digested by EcoRI and used as a template. The DNA fragment (-812 to -268) was amplified by PCR, run on an agarose gel, and stained with ethidium bromide. Lane M, DNA marker. Lane P, As a control, PCR products from the pBluescript-{lambda} II-1 clone, a plasmid containing the human NIS gene, was amplified. The size of the PCR product is 562 bp due to adapter sequences of primers.

 
Alteration of chromatin structure with histone deacetylase inhibitor
Acetylation of the amino termini of core histone can alter nucleosomal structure and has a role in facilitating the activity of sequence-specific transcription factors (37, 38). Regulation of cell growth and the activation of some specific genes generally has been associated with histone hyperacetylation (39, 40). The BHP cells do not express some thyroid-specific molecules, such as TSH receptor, TPO, or NIS. This absent expression might be due to hypoacetylation of histone around these genes. Trichostatin A (TSA), a histone deacetylase inhibitor, increases acetylated histones in many cell types (40). To investigate whether the histone hypoacetylation around the NIS gene reduces NIS gene expression in BHP cells, we performed iodide uptake assay and Northern analysis using TSA-treated BHP cells. TSA treatment did not increase iodide accumulation by BHP cells. Northern blot analysis showed no NIS mRNA expression as a consequence of the treatment (data not shown). These results suggest that the reduced NIS expression in BHP 2–7 cells is not associated with histone deacetylation. It has been reported, based on differential display, that the expression of approximately 2% of genes differ as a consequence of TSA treatment (41).

Effects of hypermethylation on NIS promoter activity
Expression of many tissue-specific genes is regulated by cytidine methylation of regulatory sequences near the transcription start site (42). Mammalian DNA methylase acts on the CG dinucleotide sequence, called a CG island, and changes the CG to 5-meCG. The prevalence of aberrant methylation pattern of some selected genes in thyroid tumors is high (43). Because the NIS promoter has 8 CG sites between -812 and the TATA box, it is possible that hypermethylation affects the NIS promoter activity. We used the demethylating agent 5-azacytidine (5-AZA) for in vivo demethylation of the promoter. 5-AZA treatment did not increase iodide accumulation in BHP cells (data not shown). Northern blot analysis showed that the treatment of BHP 2–7 and 18–21 cells with 5-AZA did not increase the level of NIS mRNA (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been reported that follicle structure or cell polarity is required for differentiated thyroid function, especially transcellular iodide transport in primary cultures of human thyroid cells (44, 45, 46, 47). We recently found that iodide uptake in long-term cultured human primary thyroid cells (18) is reduced in the monolayer condition compared with that in follicle-forming condition (21). The full expression of NIS activity in thyroid culture cells may partly involve posttranscriptional events, such as intracellular sorting, membrane localization of NIS, or another NIS regulatory factor, as suggested by several groups (12, 27, 48). Our present data, however, showed that the induction of three dimensional structure did not induce iodide uptake in BHP cell lines, although the long-term cultured human normal thyroid cells took up iodide under the same conditions.

We demonstrated reduced NIS mRNA expression in the BHP thyroid papillary cancer cell lines. This is in agreement with the data from several groups studying cancer tissue by RT-PCR (4, 5, 6, 7, 49) or immunohistochemistry (49, 50, 51, 52, 53). In contrast, increased expression of NIS in some papillary cancer tissues has been reported using Northern blot analysis and immunohistochemistry (27). It is possible that the discrepancy of NIS mRNA expression levels between the previous report (27) and the present data are due to the difference of the condition of the specimen, tissue vs. cultured cell line, or the nature of the thyroid cancer studied.

Constitutive expression of NIS in BHP cells, in which basal NIS mRNA levels were low as described above, restored iodide uptake. Thus, we concluded that reduced iodide uptake in BHP cells is the result of reduced NIS mRNA expression. We evaluated human NIS promoter activity in BHP cells compared with long-term cultured human normal thyroid cells, which take up iodide when induced into follicles (21). The sequence in the region of -596 to -268 is essential to confer full promoter activity, and this is consistent with the data reported previously (14, 15). On the other hand, the luciferase constructs with NIS promoter transfected in the BHP cell lines expressed significantly less (12–40%) luciferase activity compared with that in normal human thyroid cells. It has been reported that the region from -291 to -134 is considered as the minimal promoter of the rat NIS gene (9), and that TTF-1 binds the sequence between -245 and -230 in a cell-specific manner, and increases NIS promoter activity (9). In the human NIS promoter, however, there is no consensus element for TTF-1 between -596 and -415. Our cotransfection study of TTF-1 expression vector and p{Delta}729-Luc construct indicated that the cotransfection of TTF-1 does not increase NIS promoter activity.

Although there are two putative Pax-8 cis elements in the region between -596 and -415 (16), cotransfection of the Pax-8 expression vector did not significantly enhance expression of the -729 to -268 human NIS promoter in either normal thyroid cells or BHP cells. Pax-8 mRNA has been demonstrated in all four BHP cell lines using Northern blot analysis (17), and we confirmed the binding of endogenous Pax-8 in BHP cells to synthetic oligonucleotide of the rat NIS enhancer containing Pax-8 element. Pax-8 levels appeared to be greater in BHP 2–7 than FRTL-5 cells. Retarded bands were observed by EMSA with oligonucleotides spanning the -596 to -415 sequence and nuclear extracts from BHP cells and FRTL-5 cells. The competition study with oligonucleotides containing Pax-8 elements, however, did not demonstrate Pax-8 binding to the NIS promoter sequence. These observations demonstrated that the two putative Pax-8 elements on the NIS promoter between -596 to -415 are not used for binding to Pax-8 in these cells, and that Pax-8 is not sufficient to activate the NIS promoter in BHP cells.

Nuclear extracts from BHP 2–7 cells had reduced (probe C) or absent (probe A) binding to regions of the NIS promoter shown to be critical for expression compared with nuclear extracts from FRTL-5 rat thyroid cells. The expression of the p{Delta}596-Luc construct was markedly reduced in BHP cells compared with normal human thyroid cells and FRTL-5 cells. Therefore, the reduced or absent binding of nuclear proteins to the NIS promoter is likely to be responsible for the reduced NIS promoter activity in BHP cells. It is possible that nuclear extracts from rat-derived thyroid cells may differ in transcription factor contents from normal human thyroid, which was not tested. Our competition studies indicate that these bound nuclear proteins are not known thyroid transcription factors, such as Pax-8 or TTF-1. The augumentation of TPO promoter expression by TTF-1 and Pax-8 in BHP cells suggest that general transcription repressors are not likely to be responsible for reduced NIS expression. Recently, it has been reported that NIS gene expression in various thyroid cancer tissues determined by RT-PCR and immunohistochemistry was more closely correlated with TTF-1 compared with Pax-8 gene expression (49). In another study cotransfected Pax-8 or TTF-1 only modestly stimulated the human NIS promoter in HeLa and Cos-7 cells (54).

It has been reported that TSH/cAMP-induced up-regulation of the rat NIS gene expression requires a thyroid transcription factor, NTF-1, which also appears to be involved in TTF-1-mediated thyroid-specific NIS gene expression (10). Two possible NTF-1 sites with a consensus sequence, GNNCGGANG (10), are located -558 to -550 (1 base mismatch) (probe A) and -439 to -430 (2 base mismatch) (probe D). This sequence may be associated with up-regulation of NIS by TSH. Our DNA-nuclear protein binding study, however, showed that the rat NTF-1 element did not compete with probe A or D, which contains putative NTF-1 elements, for binding to nuclear factors from either BHP 2–7 or FRTL-5 cells.

Recently, it has been reported that treatment of KAT papillary thyroid cancer cell lines with 5-AZA, a demethylation agent, restores the iodide uptake in these cells (55). Treatment of 5-AZA, however, did not affect NIS mRNA or iodide uptake in BHP cells. KAT cell lines may be better differentiated and express the transcription factors we have detected. Reversal of hypermethylation on the NIS promoter then promotes expression of NIS.

Direct sequencing of the NIS promoter in BHP cells using the NIS gene from BHP cells as a template showed no deletion or mutations, and Northern blot analysis with TSA-treated BHP cells demonstrated that histone hypoacetylation does not explain the reduced NIS mRNA expression in BHP cells. Hypermethylation of the NIS promoter is also not sufficient to explain reduced NIS expression in BHP cells.

In conclusion, we demonstrated that reduced iodide uptake in some papillary thyroid cancer cell lines is due to reduced NIS gene expression. The absence or deficiency of unknown transcription factor(s) is involved in the reduced NIS expression. Further investigation of these nuclear factors will contribute to better understanding of the pathophysiology of thyroid cancer.


   Acknowledgments
 
We are grateful to Dr. Grady F. Saunders (Texas Medical Center, Houston, TX) for the human Pax-8 expression vector and Dr. Shioko Kimura (National Cancer Institute, Bethesda, MD) for the human TPO promoter-luciferase construct. We also thank Drs. Yoko Kanamoto and Katsumi Taki (VA Greater Los Angeles Healthcare System, CA) for technical assistance and discussions.


   Footnotes
 
This work was supported by grants from VA Medical Research Funds, VA Research Enhancement Award Program in Cancer Gene Medicine, Abbott Laboratories, and Thomas B. Rosenberg.

Abbreviations: CMV, Cytomegalovirus; DTT, dithiothreitol; NIS, sodium/iodide symporter; NTF-1, NIS TSH-responsive factor-1; TPO, thyroperoxidase; TCA, trichostatin A; TTF-1, thyroid transcription factor-1.

Received November 27, 2000.

Accepted for publication April 30, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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