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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 5 2344-2350
Copyright © 2004 by The Endocrine Society

Application of the Cre/loxP System to Enhance Thyroid-Targeted Expression of Sodium/Iodide Symporter

Xiaoqin Lin, Andrew H. Fischer, Kwon-yul Ryu, Je-yoel Cho, Thomas J. Sferra, Richard T. Kloos, Ernest L. Mazzaferri and Sissy M. Jhiang

Biochemistry Program (X.L., K.-Y.R., S.M.J.), Department of Physiology and Cell Biology (X.L., K.-Y.R., J.-Y.C., S.M.J.), Department of Pediatrics, College of Medicine (T.J.S.), Department of Internal Medicine and Radiology, Divisions of Endocrinology, Diabetes, and Metabolism, and Nuclear Medicine (R.T.K.); Center for Health Outcome Policy Evaluation Studies (E.L.M.), Ohio State University, Columbus, Ohio 43210; Department of Pathology (A.H.F.), University of Massachusetts, Worcester, Massachusetts 01655; Department of Internal Medicine, University of Florida (E.L.M.), Gainesville, Florida 32608-4653; Department of Biological Science (K.-Y.R.), Stanford University, Stanford, California 94305; and Department of Biochemistry (J.-Y.C.), School of Dentistry, Kyungpook National University, Daegu, 700-422, Republic of Korea

Address all correspondence and requests for reprints to: Sissy M. Jhiang, Ph.D., The Ohio State University, Department of Physiology and Cell Biology, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, Ohio 43210. E-mail: Jhiang.1{at}osu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Radioiodide uptake activity mediated by the human Na+/I– symporter (hNIS) in thyroid follicular cells is the basis for effective 131I therapy in thyroid cancer. However, radioiodide therapy is not effective in patients with thyroid cancer displaying low or absent hNIS expression. This study assessed the Cre/loxP system for enhancing thyroid-targeted hNIS expression driven by the thyroglobulin (Tg) promoter. The following three recombinant adenoviruses (rAd) were constructed: rAd-Tg-hNIS drives hNIS expression by the Tg promoter; rAd-Tg-Cre drives Cre expression by the Tg promoter; and rAd-CMV-loxP-hNIS drives hNIS expression by the cytomegalovirus (CMV) promoter after Cre-mediated excision of an intervening loxP-GFP-Zeo-loxP. Immortalized normal and malignant rat thyroid cell lines and primary cultures of normal human thyroid and human follicular adenoma cells were investigated. We found that the relative promoter activity of Tg vs. CMV is critical for the efficacy of the Cre/loxP system. In cells with weak Tg promoter activity, coinfection of rAd-Tg-Cre and rAd-CMV-loxP-hNIS induced higher hNIS expression than single infection of rAd-Tg-hNIS. Finally, Tg promoter activity was partially restored in malignant thyroid cells by forced expression of the paired domain-containing transcription factor (Pax-8), allowing the Cre/loxP system to mildly enhance radioiodide uptake.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID CARCINOMA IS the most common endocrine malignancy, including differentiated thyroid carcinoma (DTC) and undifferentiated (anaplastic) thyroid carcinoma (1). DTC (papillary and follicular carcinoma) comprises about 85–90% of all thyroid malignancies (2). Most patients with DTC can be effectively treated with surgery followed by radioiodide-131 ablation and have a relatively good prognosis (3, 4). However, approximately one third of patients with DTC and all patients with anaplastic carcinoma cannot benefit from radioiodine therapy due to reduced or absent ability to concentrate iodide (2, 5). It has been reported that the loss of radioiodine accumulation in thyroid cancer is due, in part, to decreased expression of the Na+/I– symporter (NIS), the key molecule that mediates the active uptake of iodide into thyroid tissues (6, 7, 8, 9). Thus, NIS gene transfer to restore and/or increase radioiodide uptake (RAIU) activity represents a promising means to improve the effectiveness of radioiodine therapy in patients with non-iodine-avid thyroid cancers. The objective of this study is to investigate the efficacy of transcriptionally targeted NIS expression in a variety of thyroid cells.

The thyroglobulin (Tg) promoter has been extensively studied for its potential use in transcriptionally targeted thymidine kinase suicide gene therapy of thyroid cancer (10, 11, 12). The Tg promoter is an ideal candidate to confer thyroid-targeted gene expression due to its high level of thyroid-specific transcriptional activity. Unfortunately, it has been reported that the Tg promoter activity is weaker in thyroid carcinomas than in normal thyroid tissues (13). A possible way to enhance Tg promoter-driven gene expression in thyroid carcinomas is the application of the Cre/loxP system. Several reports have demonstrated the ability of the Cre/loxP system to enhance transcriptionally targeted suicide gene expression using the Tg promoter or the carcinoembryonic antigen promoter in thyroid or gastrointestinal cancer cells, respectively (11, 14, 15, 16, 17).

In this paper, we investigated and compared two different adenoviral systems in providing thyroid-specific human NIS (hNIS) expression (Fig. 1AGo). The first system involves a single adenovirus, in which hNIS expression is driven by the Tg promoter (rAd-Tg-hNIS). The second system involves two adenoviruses, one in which the Tg promoter drives Cre recombinase expression (rAd-Tg-Cre) and another in which the cytomegalovirus (CMV) promoter is separated from the hNIS gene by two loxP (Cre recombinase recognition) sites (rAd-CMV-loxP-hNIS). Coinfection of these two adenoviruses into thyroid cells results in Cre expression, which then excises an intervening DNA sequence bearing a stop codon between the two loxP sites, thereby allowing hNIS expression under control of the strong CMV promoter (Fig. 1BGo). Here, we show that the relative promoter activity of Tg vs. CMV is critical for the ability of the Cre/loxP system to enhance thyroid-targeted hNIS expression. Thus, in cells with weak Tg promoter activity, coinfection of rAd-Tg-Cre and rAd-CMV-loxP-hNIS induced higher hNIS expression than single infection of rAd-Tg-hNIS.



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  		FIG. 1. A, Schematic representation of the E1/E3-deleted adenovirus vector constructs. rAd-Tg-hNIS and rAd-Tg-Cre express hNIS gene and Cre gene, respectively, under the control of the Tg promoter. rAd-CMV-loxP-hNIS is designed to express hNIS gene under the strong CMV promoter only after Cre recombinase excises the GFP-Zeo fusion gene. The ends of the Zeo and hNIS genes both contain a stop codon (*). B, Increased hNIS expression will be induced in cells with an active Tg promoter by the Tg-Cre/CMV-loxP-hNIS system. Cre recombinase excises GFP-Zeo fusion gene between the two loxP sites as a covalently closed circle, leaving one loxP site behind and allowing transcription of hNIS driven by the strong CMV promoter.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and stable clones

Cos-7 monkey kidney cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). FRTL-5 differentiated normal rat thyroid cells (kindly provided by Dr. Leonard D. Kohn, Ohio University, Athens, OH) were cultured in Coon’s modified Ham’s F-12 medium, supplemented with 5% calf serum and six hormone (6H) mixture as described previously (18). Malignant FRTC cells were derived from FRTL-5 cells transformed with muramyl dipeptide (kindly provided by Dr. Makoto Iitaka, Saitama Medical School, Saitama, Japan) and maintained in the same medium as FRTL-5 cells.

Malignant FRTC cells were stably transfected with 5 µg rat paired domain-containing transcription factor (Pax-8)/pcDNA3 (our unpublished data) by calcium phosphate transfection. The DNA precipitates were replaced with fresh medium 6 h later. FRTC/Pax-8 cells were selected with 320 µg/ml of geneticin (G-418) 48 h posttransfection. The selected clones were then screened for the level of Pax-8 protein by Western blot analysis (data not shown).

Preparation of human normal or tumor primary thyroid culture

Human normal thyroid tissue and human follicular adenoma tissue were put into tissue culture as described previously with minor modification (19). Fresh tissue (0.1–1 g) was cut into approximately 1-mm cubes and washed with cold Hanks’ Balanced Salt Solution (HBSS; calcium and magnesium free). The tissue was then digested with digestion solution [fresh clostridium collagenase (100 U/ml) and Dispase (1 mg/ml) in HBSS] for 1.5 h at 37 C with 5% CO2 and agitated with a large bore pipette every 30 min. The large undigested fragments were allowed to settle briefly, and the supernatant containing released epithelial clusters was transferred to a fresh flask with the addition of 10% FBS and maintained at 37 C. The remaining tissue fragments continued to be digested by incubating with fresh digestion solution at 37 C until the digested tissue became very soft and filmy. This entire sample and the previously collected supernatant were filtered through a 70-mm falcon cell strainer to remove the remaining clumps that consist largely of connective tissue. The cells were then washed with HBSS three times and cultured in a 2:1:1 mixture of DMEM, Ham’s F-12, and MCDB-104 (Life Technologies, Inc., Paisley, UK) supplemented with 10% FBS. Human Investigation Committee approval for these studies was obtained at the University of Massachusetts (Institutional Review Board no. H-10396).

Construction and production of rAd

The plasmid pSh-Tg-hNIS was constructed as follows. A KpnI-PmeI fragment containing the hNIS gene was excised from pSh-CMV-hNIS (20) and cloned into the pShuttle vector. The 2.0-kb bovine Tg promoter (located from –2036 to +9 nucleotide relative to the transcriptional start site) was then released as a SpeI-KpnI fragment from pSh-Tg-PTC3 and inserted upstream of the hNIS gene to generate pSh-Tg-hNIS.

The plasmid pSh-Tg-Cre was constructed as follows. The Cre recombinase gene and 588 bp of polyA sequence from the PBS185 vector were released as a SpeI (blunt-ended by T4 DNA polymerase)-XbaI fragment and replaced the hNIS gene in pSh-Tg-hNIS. The extra 588-bp polyA fragment was then removed by MluI/XbaI digestion to generate pSh-Tg-Cre.

The plasmid pSh-CMV-loxP-hNIS was constructed as follows. The loxP-GFP-Zeo-loxP fragment from the pCMV-ß-4-REV vector was subcloned as a NotI (blunt-ended by T4 DNA polymerase) fragment into the KpnI site of pSh-CMV-hNIS.

rAd-Tg-hNIS, rAd-Tg-Cre, and rAd-CMV-loxP-hNIS were produced and purified as described previously (20, 21). The titers of adenoviruses were determined by plaque formation in 293 cells and expressed in pfu/ml.

Detection of hNIS protein by Western blot analysis

Cells were plated in 100-mm dishes. When cells became 70–80% confluent, they were infected with various adenoviruses at the indicated multiplicity of infection (MOI) for 2 h. Forty-eight hours postinfection, cells were harvested for Western blot analysis as described previously with minor modification (22). Briefly, the membrane fractions of the cells were subjected to 7.5% SDS-PAGE. The proteins were transferred to nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane was incubated with the affinity-purified hNIS antibody (1:1500), which recognizes the COOH terminus of hNIS protein, for 1 h at room temperature, followed by incubation with horseradish peroxidase-conjugated donkey {alpha}-rabbit IgG (1:4000) (Amersham Pharmacia, Piscataway, NJ) for 1 h at room temperature. The filter was incubated with the enhanced chemiluminescence detection reagents (Amersham) for 1 min and exposed to x-ray film.

Detection of NIS function by RAIU assay

Cells were seeded in 24-well plates. When cells became 70–80% confluent, they were incubated with various adenoviruses at the indicated MOI for 2 h. Forty-eight hours postinfection, RAIU assay was performed as described previously (20). Briefly, cells were incubated with 2 µCi Na 125I for 30 min at 37 C with 5% CO2. Cells were then washed with cold HBSS twice, and the radioiodide taken up by the cells was released by 95% ethanol for 20 min at room temperature. The radioactivity was counted by a {gamma}-counter (Packard Instruments, Downers Grove, IL). Experiments were performed in triplicate.

Detection of the promoter activities of CMV and Tg by luciferase assay

Cells were seeded in six-well plate. When cells became 50–60% confluent, calcium phosphate transfection or FuGENE 6 transfection (Roche, Nutley, NJ) was performed using 3 µg of Tg/pGL2B (18) or CMV/pGL2B (our unpublished data), in which Tg or CMV promoter was inserted upstream of the promoterless luciferase reporter gene in pGL2B vector, respectively. CMV/ß-galactosidase DNA construct (0.2 µg) was cotransfected with each DNA construct to normalize the transfection efficiency. The DNA precipitates were replaced with fresh medium 6 h later for calcium phosphate transfection. Forty-eight hours posttransfection, the cells were lysed with 80 µl lysis buffer provided in the luciferase assay kit (Promega Corporation, Madison, WI). The luciferase and ß-galactosidase assays were carried out as described previously (18). Experiments were performed in duplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of functional rAds: rAd-Tg-hNIS, rAd-Tg-Cre, and rAd-CMV-loxP-hNIS

Three rAds, rAd-Tg-hNIS, rAd-Tg-Cre, and rAd-CMV-loxP-hNIS, were constructed as described in Materials and Methods. To verify the function of these three rAds, various adenoviruses were infected into FRTL-5 rat normal thyroid cells or Cos-7 monkey kidney cells. The level of hNIS protein in infected cells was detected by Western blot analysis. As expected, rAd-Tg-hNIS can only express hNIS protein in Tg-expressing FRTL-5 cells but not in Cos-7 cells (Fig. 2AGo). Similarly, hNIS expression was detected only in FRTL-5 cells but not in Cos-7 cells after coinfection with rAd-Tg-Cre and rAd-CMV-loxP-hNIS (Fig. 2BGo). The cells infected with rAd-CMV-loxP-hNIS alone did not induce hNIS expression, indicating that Cre recombinase is required for hNIS expression in the dual virus system (Fig. 2B). It is notable that although FRTL-5 cells express endogenous rat NIS (rNIS), our hNIS antibodies do not cross-react with rNIS (see lane 3 in Fig. 2BGo).



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  		FIG. 2. The three adenoviruses were functional in cultured cells by Western blot analysis of hNIS protein. A, rAd-Tg-hNIS (MOI = 10) conferred hNIS expression only in differentiated FRTL-5 thyroid cells that express Tg but not in Cos-7 monkey kidney cells. The figure shown was taken with 3 min of exposure. B, Coinfection of rAd-Tg-Cre and rAd-CMV-loxP-hNIS (MOI = 10:10) only induced hNIS expression in FRTL-5 cells but not in Cos-7 cells. rAd-CMV-loxP-hNIS (MOI = 20) alone did not induce hNIS expression in FRTL-5 cells, indicating that Cre recombinase is required for hNIS expression in the dual virus system. The figure shown was taken with 10 sec of exposure. Five-microgram membrane fractions were loaded for each lane. Note the mature hNIS glycoprotein (90 kDa) and the partially glycosylated hNIS protein (60 kDa). MW, Molecular weight.

 
Enhanced hNIS expression and RAIU activity in FRTL-5 rat normal thyroid cells coinfected with rAd-Tg-Cre and rAd-CMV-loxP-hNIS

To compare the abilities of the Tg-hNIS and Tg-Cre/CMV-loxP-hNIS systems to mediate hNIS expression and function, we infected FRTL-5 cells with either rAd-Tg-hNIS alone (MOI = 20) or with both rAd-Tg-Cre and rAd-CMV-loxP-hNIS (MOI = 10:10). Analysis of coinfection with the Cre/loxP system revealed that optimal hNIS expression occurred when cells were administered with a 1:1 ratio of rAd-Tg-Cre vs. rAd-CMV-loxP-hNIS (data not shown). Because hNIS antibodies do not detect endogenous rNIS in FRTL-5 cells, we were able to compare the expression levels of hNIS induced by the designated adenovirus. Our data showed that both Tg-hNIS and Tg-Cre/CMV-loxP-hNIS induced hNIS expression in FRTL-5 cells; however, the level of hNIS protein in FRTL-5 cells coinfected with the Tg-Cre/CMV-loxP-hNIS system was significantly higher than that in cells infected with rAd-Tg-hNIS alone (Fig. 3AGo). It is difficult to quantify the relative hNIS expression levels induced by one system vs. another because different exposure times were required to optimize the detection of hNIS protein.



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  		FIG. 3. Coinfection of rAd-Tg-Cre and rAd-CMV-loxP-hNIS (MOI = 10:10) enhanced hNIS expression and RAIU activity in FRTL-5 cells compared with single infection of rAd-Tg-hNIS (MOI = 20). A, Western blot analysis of hNIS expression (0.5 µg per lane). The hNIS expression level in cells coinfected with rAd-Tg-Cre/rAd-CMV-loxP-hNIS was much higher than that in cells infected with rAd-Tg-hNIS. Thus, different exposure times were required to optimize the detection of hNIS protein induced by the Tg-hNIS and Tg-Cre/CMV-loxP-hNIS systems. The figure shown was taken with 1 min of exposure. MW, Molecular weight. B, RAIU assay for NIS function (the sum of endogenous rNIS and exogenous hNIS activities). The noninfected FRTL-5 cells served as a control. The same passage of FRTL-5 cells (P23) was used for Western blot analysis and RAIU assay.

 
As shown in Fig. 3BGo, the RAIU activity in FRTL-5 cells was significantly increased by infection of rAd-Tg-hNIS alone as well as by coinfection of rAd-Tg-Cre and rAd-CMV-loxP-hNIS. Interestingly, although Tg-Cre/CMV-loxP-hNIS is much more efficient than Tg-hNIS in inducing hNIS expression, the increase of RAIU activity in cells coinfected with the Tg-Cre/CMV-loxP-hNIS system was only slightly higher than that in cells infected with rAd-Tg-hNIS alone (Fig. 3BGo). Thus, it appears that not all induced hNIS resulted in increasing RAIU activity in infected FRTL-5 cells. This may be because noninfected FRTL-5 cells already have high-level iodide uptake due to endogenous rNIS (see Discussion).

Enhanced hNIS expression and RAIU activity in FRTC/Pax-8 rat malignant thyroid cells coinfected with rAd-Tg-Cre and rAd-CMV-loxP-hNIS

Malignant FRTC thyroid cells are reported to express low levels of Tg mRNA (10, 23), which is similar to less differentiated thyroid tumors. However, hNIS expression was not detectable by Western blot analysis in malignant FRTC cells infected with either rAd-Tg-hNIS alone or rAd-Tg-Cre and rAd-CMV-loxP-hNIS (data not shown). Our luciferase assay showed that malignant FRTC cells had extremely weak, if any, Tg promoter activity (data not shown). It has been reported that the reintroduction of a thyroid-specific transcription factor, Pax-8, into PCPy cells, which are derived from the differentiated PC Cl 3 immortalized rat thyroid cells transformed with polyoma middle T antigen, was sufficient to increase Tg promoter activity (24). Therefore, we stably transfected rat Pax-8 into malignant FRTC cells (FRTC/Pax-8) to increase Tg promoter activity. As shown in Fig. 4AGo, hNIS expression in FRTC/Pax-8 cells coinfected with rAd-Tg-Cre/rAd-CMV-loxP-hNIS (MOI = 20:20) was higher than that of cells infected with rAd-Tg-hNIS alone (MOI = 40). In cells infected with the Cre/loxP system (MOI = 25:25), the RAIU mediated by exogenous hNIS was higher than in cells infected with rAd-Tg-hNIS (MOI = 50; Fig. 4BGo). We also observed a slight increase in background RAIU in FRTC stably transfected with Pax-8 compared with FRTC parental cells (data not shown). This increase may be due to the direct stimulating effect of Pax-8 on the endogenous rNIS promoter. It is noteworthy that the RAIU activity in FRTC/Pax-8 cells (Fig. 4BGo) is much lower than that in FRTL-5 cells (Fig. 3BGo).



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  		FIG. 4. Coinfection of rAd-Tg-Cre and rAd-CMV-loxP-hNIS enhanced hNIS expression and RAIU activity in FRTC malignant thyroid cells stably transfected with Pax-8 compared with single infection with rAd-Tg-hNIS. A, Western blot analysis of hNIS expression in cells either infected with rAd-Tg-hNIS (MOI = 40) or coinfected with rAd-Tg-Cre and rAd-CMV-loxP-hNIS (MOI = 20:20; 100 µg per lane). The figure shown was taken with 2 min of exposure. MW, Molecular weight. B, RAIU assay for NIS function in cells either infected with rAd-Tg-hNIS (MOI = 50) or coinfected with rAd-Tg-Cre and rAd-CMV-loxP-hNIS (MOI = 25:25). Noninfected FRTC/Pax-8 cells served as a control.

 
Enhanced RAIU activity in normal human thyroid primary culture coinfected with rAd-Tg-Cre and rAd-CMV-loxP-hNIS

Short-term primary cultures of normal human thyrocytes serve as a physiologic in vitro model. To evaluate the efficacy of the Tg-Cre/CMV-loxP-hNIS system in the primary culture of human normal thyrocytes, cells were either infected with rAd-Tg-hNIS alone (MOI = 20) or coinfected with rAd-Tg-Cre and rAd-CMV-loxP-hNIS (MOI = 10:10). Only RAIU assay was performed to investigate hNIS function due to the availability of cell numbers. As shown in Fig. 5Go, the RAIU activity was increased by both systems, and the increase of RAIU activity in cells coinfected with Tg-Cre/CMV-loxP-hNIS was significantly higher than that in cells infected with rAd-Tg-hNIS alone. Similarly to FRTL-5 cells, the primary culture infected with control adenovirus displayed a relatively high level of iodide uptake, which is due to the expression of endogenous hNIS protein in the normal primary culture.



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  		FIG. 5. Coinfection of rAd-Tg-Cre and rAd-CMV-loxP-hNIS (MOI = 10:10) significantly enhanced RAIU activity in normal human thyroid primary culture compared with single infection of rAd-Tg-hNIS (MOI = 20). Cells infected with rAd-CMV-loxP-hNIS (MOI = 20) alone served as a control for endogenous hNIS.

 
Enhanced hNIS expression and RAIU activity in human thyroid follicular adenoma primary culture coinfected with rAd-Tg-Cre and rAd-CMV-loxP-hNIS

A human thyroid follicular adenoma primary culture was derived from a thyroid follicular adenoma that was cold on the radioiodide scan 3 months before surgery, which implies the absent or weak hNIS expression or function in the cells compared with surrounding normal cells. To reintroduce hNIS expression and evaluate the efficacy of the Tg-Cre/CMV-loxP-hNIS system in this follicular adenoma, cultured cells were either infected with rAd-Tg-hNIS alone (MOI = 20) or coinfected with rAd-Tg-Cre and rAd-CMV-loxP-hNIS (MOI = 10:10). Western blot analysis and RAIU assay were performed to examine hNIS expression and hNIS function. Again, as shown in Fig. 6Go, hNIS expression levels and the increase of RAIU activity induced by Tg-Cre/CMV-loxP-hNIS (MOI = 10:10) were significantly higher than those induced by rAd-Tg-hNIS alone (MOI = 20). The cells infected with rAd-CMV-loxP-hNIS alone (MOI = 20) showed low RAIU activity.



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  		FIG. 6. Coinfection of rAd-Tg-Cre and rAd-CMV-loxP-hNIS (MOI = 10:10) enhanced hNIS expression and RAIU activity in human thyroid follicular adenoma primary culture compared with single infection of rAd-Tg-hNIS (MOI = 20). A, Western blot analysis of hNIS expression (30 µg per lane). The figure shown was taken with 2 min of exposure. MW, Molecular weight. B, RAIU assay for NIS function. Cultured cells infected with rAd-CMV-loxP-hNIS (MOI = 20) alone served as a control.

 
Relative promoter activity of Tg vs. CMV in targeted thyroid cells crucial for the efficacy of the Tg-Cre/CMV-loxP-hNIS system

In this paper, we hypothesize that the Tg-Cre/CMV-loxP-hNIS system is able to enhance thyroid-targeted hNIS expression. The hypothesis is mainly based on the assumption that CMV promoter activity is much stronger than Tg promoter activity in targeted cells. Thus, the relative promoter activity of Tg vs. CMV in the targeted cells will be vital for the efficacy of Tg-Cre/CMV-loxP-hNIS. Experiments were performed to correlate the relative promoter activity of Tg vs. CMV with the efficacy of Tg-Cre/CMV-loxP-hNIS in targeted thyroid cells. The investigated cells were divided into four experimental groups. Cells were either transfected with Tg/pGL2B or CMV/pGL2B to determine Tg or CMV promoter activity by luciferase assay. Simultaneously, cells were either infected with rAd-Tg-hNIS alone (MOI = 20) or coinfected with the Tg-Cre/CMV-loxP-hNIS system (MOI = 10:10) to determine RAIU activity. As shown in Fig. 7Go, when the relative promoter activity of Tg vs. CMV is 1:5 in P14 FRTL-5 cells, Cre/loxP is not effective to further enhance Tg-targeted hNIS-mediated RAIU activity. In comparison, when Tg promoter activity was much weaker than CMV promoter activity in P20 FRTL-5 (the relative promoter activity of Tg vs. CMV is 1:333), the Cre/loxP system was more effective in increasing RAIU activity than Tg-hNIS alone. In primary cultures of human thyrocytes, in which the relative promoter activity of Tg vs. CMV was about 1:1600 or 1:1500, the Tg-Cre/CMV-loxP-hNIS system was very effective in enhancing Tg-targeted hNIS-mediated RAIU activity (Fig. 7Go). In addition, it was interesting to note that the CMV promoter activity varied among different cells (data not shown).



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  		FIG. 7. Relative promoter activity of Tg vs. CMV in targeted thyroid cells is crucial for the efficacy of the Tg-Cre/CMV-loxP-hNIS system. (A) Relative promoter activity of Tg vs. CMV in P14 FRTL-5 cells, P20 FRTL-5 cells, normal human thyroid primary culture, and human thyroid follicular adenoma primary culture. Tg/pGL2B or CMV/pGL2B was transfected into targeted thyroid cells separately to analyze Tg promoter and CMV promoter activity, respectively. The luciferase activity of CMV/pGL2B was reported relative to the luciferase activity of Tg/pGL2B, which was arbitrarily designated as 1. All experiments were performed in duplicate. (B) RAIU assay for NIS function in targeted thyroid cells either infected with rAd-CMV-loxP-hNIS (MOI = 20) alone, rAd-Tg-hNIS (MOI = 20) alone, or coinfected with rAd-Tg-Cre and rAd-CMV-loxP-hNIS (MOI = 10:10).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several groups have investigated the potential of NIS-mediated radionuclide imaging and therapy in thyroid or nonthyroidal cancer cells (5, 25, 26). However, most studies express NIS under the control of the strong and constitutive CMV promoter as proof-of-principle experiments. To apply NIS-based gene therapy to patients, it is important to restrict NIS expression to targeted cells to avoid unwanted side effects in nontargeted tissues. For patients after total thyroidectomy, NIS expression may be restricted to residual thyroid cancer using Tg as a tissue-specific promoter. However, Tg promoter activity is generally much weaker than that of a constitutive viral promoter. Thus, the Tg-Cre/CMV-loxP-hNIS system may be superior to a Tg-hNIS system to induce hNIS expression in thyroidal tissues. In this study, we showed that the Cre/loxP system was effective in enhancing transcriptionally targeted NIS expression/function in FRTL-5 cells, FRTC cells, normal human thyroid primary culture, and human thyroid follicular adenoma primary culture. In addition, we showed that the efficacy of Tg-Cre/CMV-loxP-hNIS is dependent upon the relative promoter activity of Tg vs. CMV. Thus, for thyroid cancers with strong Tg promoter activity, a Tg-hNIS system may be sufficient to confer high levels of hNIS expression to improve radioiodide therapy. For thyroid cancers with weak Tg promoter activity, Cre/loxP is an attractive system to enhance Tg-targeted NIS expression. However, for thyroid cancers with negligible Tg promoter activity, strategies to increase Tg promoter activity must also be used to confer Tg-targeted hNIS expression.

Immortalized FRTL-5 cells have been known to display different characteristics with age or transformation. Serially passaged FRTL-5 cells are reported to dedifferentiate and lose Tg expression (27). This is consistent with our data that the aged P20 FRTL-5 cells had weaker Tg promoter activity than the young P14 FRTL-5 cells (Fig. 7AGo). Similarly, decreased Tg promoter activity has been reported in FRTC tumor of the 12th generation compared with the third generation (23). Although forced expression of Pax-8 in FRTC cells increased Tg promoter activity and allowed Tg-targeted hNIS expression/function, the increase of RAIU activity in FRTC/Pax-8 cells coinfected with Tg-Cre/CMV-loxP-hNIS was not high enough to justify further animal study. We also evaluated the effects of Cre/loxP system on FTC133 human thyroid carcinoma cells (28). However, FTC133 cells exhibited undetectable Tg promoter activities and did not induce RAIU activity by the Tg-hNIS system or the Cre/loxP system (data not shown). Therefore, a non-iodine-avid tumorigenic thyroid tumor cell line, yet with some retained Tg promoter activity, will be essential to examine the efficacy of the Cre-loxP system to increase Tg-targeted hNIS expression/function in preclinical animal models.

It is interesting to note that the extent of increased RAIU activity does not appear to be proportional to the extent of induced hNIS expression in all cells studied. For cells with high levels of endogenous NIS expression and function, it is possible that the cell surface is eventually saturated with functional NIS, excluding further cell surface trafficking of exogenous hNIS. However, for cells with low or absent endogenous NIS, defects in cell surface trafficking of hNIS are suspected. The proper and efficient cell surface trafficking has been shown to play an important role in the functional expression of hNIS in thyroid cells (9, 29, 30). Thus, possible defects in cell surface trafficking of hNIS in malignant cells remain a real challenge for hNIS-based gene therapy.

To apply Tg-targeted NIS gene therapy, Tg promoter activity in cancer cells needs to be sufficient, if not maximized. In this study, we showed that forced expression of Pax-8 into FRTC cells slightly increased Tg promoter activity and induced detectable hNIS expression. Thyroid transcription factor 1 (TTF-1) has also been shown to reactivate the Tg promoter in thyroid carcinoma cells (31, 32). Therefore, forced expression of both TTF-1 and Pax-8 may further facilitate the efficacy of Tg-targeted NIS gene therapy. In addition, a tandem repeat of the minimal Tg promoter has been shown to induce much higher thymidine kinase expression in transduced thyroid cells than the single Tg promoter did (33). Thus, it is desirable to generate a composite Tg promoter yielding the highest thyroid-specific promoter activity. Furthermore, the efficiency of Cre-directed site-specific recombination could be enhanced by facilitating Cre nuclear localization (34). Finally, the efficacy of the Cre/loxP system may be further improved by dually expressing Tg-Cre and CMV-loxP-hNIS in a single adenovirus to avoid inefficient coinfection of two adenoviruses.

In conclusion, the Tg-Cre/CMV-loxP-hNIS system can significantly enhance thyroid-targeted hNIS expression and induce RAIU activity in thyroid cells with weak Tg promoter activity. We showed that the relative promoter activity of Tg vs. CMV is crucial for the efficacy of the Cre/loxP system. Thus, before NIS-based gene therapy, Tg promoter activity should be evaluated, for example, by measuring serum Tg levels upon TSH stimulation in patients.


   Acknowledgments
 
We thank Dr. Leonard D. Kohn at Ohio University (Athens, OH) for providing FRTL-5 cells; Dr. Makoto Iitaka at Saitama Medical School (Saitama, Japan) for providing FRTC cells; Dr. Yuji Nagayama at Nagasaki University (Nagasaki, Japan) for helpful discussion about FRTC cells; and Mr. Derek K. Marsee for support in manuscript preparation.


   Footnotes
 
This work was supported in part by Department of Defense Prostate Cancer Research Program DAMD 17-02-0119 (to S.M.J.).

Abbreviations: CMV, Cytomegalovirus; DTC, differentiated thyroid carcinoma; FBS, fetal bovine serum; HBSS, Hanks’ balanced salt solution; MOI, multiplicity of infection; NIS, Na+/I– symporter; rAd, recombinant adenovirus; RAIU, radioiodide uptake; Tg, thyroglobulin.

Received November 12, 2003.

Accepted February 11, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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