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Original Article |
Department of Histology, Microbiology, and Medical Biotechnologies (L.B., R.B., I.C., E.G., C.P., G.P.), University of Padova, I-35121 Padova; Department of Pathology (M.C.), University of Verona, I-37134 Verona; and Department of Internal Medicine (M.B.), University of Ancona, I-60100 Ancona, Italy
Address all correspondence and requests for reprints to: Giorgio Palù, M.D., Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Via Gabelli 63, I-35121 Padova, Italy. E-mail: giorgio.palu{at}unipd.it.
Abstract
Gene therapy may be an effective approach to thyroid carcinoma refractory to conventional treatment. A transcriptionally targeted retroviral vector for gene therapy of thyroid carcinomas was generated replacing the viral enhancer with the enhancer sequence of the human thyroglobulin (TG) gene, yielding a chimeric long-terminal repeat. The TG enhancer was used to drive the expression of either a reporter gene (ß-galactosidase) or two therapeutic genes, i.e. the prodrug-activating enzyme thymidine kinase of herpes simplex virus (HSV-TK) and human IL-2, separated by an internal ribosome entry site. The corresponding vector having an unmodified long-terminal repeat was used as control. The targeted vector allowed selective transgene expression and cell killing in differentiated thyroid tumor cells but not in anaplastic thyroid carcinoma cells and nonthyroid cells, as demonstrated by quantitative RT-PCR and cytotoxicity assays. Nude mice injected with tumor cells underwent near complete or complete regression of tumors transduced with the control vector after ganciclovir treatment. On the other hand, infection with the thyroid-specific vector led to regression only of TG-expressing tumors. In addition, tumors expressing human IL-2 showed significant growth retardation, compared with nontransduced tumors while exhibiting signs of necrosis and presence of an inflammatory infiltrate. However, HSV-TK/IL-2 plus ganciclovir was significantly more efficient than HSV-TK/IL-2 alone in eradicating tumor masses. Our results indicate that replacement of viral enhancer with TG enhancer confers selectivity of transgene expression in thyroid cells. Thus, the combined thyroid-specific expression of two therapeutic genes (cytokine and suicide genes), although a safe tumor-targeted treatment, would allow an increased anticancer effect.
THYROID CARCINOMAS REPRESENT the most common endocrine malignancy, accounting for about 1% of all human cancers. Differentiated thyroid cancer generally responds to conventional therapy and has a relatively good prognosis; however, about 30% of relapsing carcinomas show an aggressive and highly malignant behavior, associated with a poor survival. Several gene therapy strategies have been designed for the treatment of thyroid carcinomas and adopted both in vitro and in vivo, including tumor suppressor gene replacement, prodrug activation, and immunotherapy (1, 2). Because thyroid carcinomas typically express tissue- and tumor-specific genes, they represent an ideal model for a targeted gene therapy approach, whereby thyroid-specific enhancer/promoter sequences drive the expression of therapeutic genes. We report here the development and characterization of a transcriptionally targeted retroviral vector obtained by reshuffling of the long-terminal repeat (LTR), i.e. by replacement of the viral enhancer in the LTR with target cell-specific enhancer elements (3, 4). Moreover, to amplify the antitumor response, the same retroviral vector construct combines two different therapeutic modalities, i.e. enzyme-directed prodrug activation (herpes simplex virus-1 thymidine kinase, HSV-TK) along with cytokine- promoted (IL-2) tumor rejection, an approach we pioneered in a pilot study to treat patients with recurrent glioblastoma multiforme (5, 6).
Materials and Methods
Construction of recombinant retroviral vectors and retroviral transduction
Recombinant retroviral vector pMFGnlsLacZ (7) was a generous gift from Dr. R. C. Mulligan (Department of Genetics, Harvard Medical School, Boston, MA). To replace retroviral enhancer with human thyroglobulin (TG) enhancer, the nlsLacZ was removed and the 3' LTR was excised from pMFGnlsLacZ vector by BamHI and EcoRI digestion and ligated into the pGEX4T3 plasmid (Promega Corp., Madison, WI). A 267-bp DNA fragment containing retroviral enhancer sequence was removed from 3' LTR U3 region by NheI and XbaI digestion and replaced by a 650-bp DNA fragment containing human TG enhancer, obtained from 1.4phTgCAT plasmid (8) (a kind gift from Dr. D. Christophe, IRIBHN, University of Brussels, Brussels, Belgium) by AflIII and StuI digestion. Chimeric LTR was subsequently excised from pGEX4T3 and ligated into pMFG in its original position. The resulting vector was named pMFG(TGenh). To generate the reporter vector pMFGnlsLacZ(TGenh), the nlsLacZ sequence was ligated into the BamHI site of pMFG(TGenh).
The pMFGIL-2TKSN(TGenh) and pMFGIL-2TKSN vectors were constructed by subcloning the IL-2TK cassette, which contains the human IL-2 (hIL-2) gene and HSV-TK gene, separated by an internal ribosome entry site sequence. The cassette, which had been excided from LIL-2TKSN (5) by EcoRI digestion, was blunt ended and ligated into BglII and NcoI sites of pMFG-TG(enh) and pMFG vectors. A 1295-bp cassette containing the neomycin-resistance gene under the control of Simian virus 40 (SV40) early promoter (SV40neo), obtained from pLXSN (9) by NheI and XhoI digestion, was blunt ended and ligated into the BamHI site of both vectors.
Plasmid vectors were transfected into the amphotropic packaging cell line FLYA13 by using the calcium phosphate transfection system (Invitrogen, Leek, the Netherlands) reagents, as described (10). Cells transfected with vectors containing SV40neo were selected in a medium containing 800 µg/ml G418 (Invitrogen) and single cell-derived clones isolated and expanded to cell lines. Viral titer was determined by infection of NIH3T3 cells with virus-containing supernatants from single cell-derived clones of FLYA13 producer cells, as described previously (10). Supernatants from producer cell clones with higher viral titer were used to transduce target thyroid and nonthyroid cells. The cells were incubated for 4 h with viral supernatants in the presence of 8 µg/ml polybrene (Sigma, St. Louis, MO) and after 48 h selected with 800 µg/ml G418 (10).
Cell lines
Four different human thyroid carcinoma cell lines [i.e. WRO and follicular thyroid carcinoma (FTC)-133 cells, established from metastases of follicular thyroid carcinoma; C8305 cells, established from a primary undifferentiated thyroid carcinoma; ARO cells, derived from anaplastic thyroid carcinoma] and four nonthyroid cell lines (human cervical carcinoma HeLa, human astrocytoma AoU373, human hepatocellular carcinoma HepG2, mouse fibroblasts NIH3T3) were used. WRO (HTL98002, Interlab Cell Line Collection, ICLC, Genova, Italy), FTC-133 (HTL97015, ICLC), C8305 (HTL96026, ICLC), HeLa [CRL-2; American Type Culture Collection (ATCC), Manassas, VA], and AoU373 (HTB-17; ATCC) cells were cultured in DMEM (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen), 100 U/ml penicillin G and 100 µg/ml streptomycin. ARO (UCLA RO-81-A-1, CA), HepG2 (HB-8065; ATCC), and NIH3T3 (CRL-1658; ATCC) cells were grown in RPMI-1640 medium (Invitrogen) supplemented with 10% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin.
The FLYA13 packaging cell line (11), a kind gift from Dr. Y. Takeuchi (Chester Beatty Laboratories, London, UK), derived from HT1080 human fibrosarcoma cells, was maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin G, 100 µg/ml streptomycin, 4 µg/ml blasticidin S (ICN Biomedicals, Inc., Aurora, OH), and 10 µg/ml phleomycin (Sigma).
In vitro LacZ gene expression with retrovirus infection
Recombinat retroviral particles were produced by transient transfection of FLYA13 packaging cells with pMFGnlsLacZ(TGenh) and pMFGnlsLacZ plasmids by DNA/calcium phosphate precipitation. Target cells were seeded at 2 x104 cells/well in 24-well culture plates either in the presence or absence of TSH 10 UI/liter (Sigma). After 48 h, the cells were infected with virus-containing supernatant from FLYA13 producer cells, in the presence of 8 µg/ml polybrene. After 48 h, the cells were stained with X-gal (5-bromo-4-chloro-indolyl-ß-D-galactophyranoside) to evaluate ß-galactosidase expression, as described (10).
RNA isolation and quantitative real-time RT-PCR analysis
Total RNA was isolated from cells following a single-step acid guanidium phenol-chloroform extraction procedure using RNAzol (Biotech Laboratories, Inc., Houston, TX). Random-primed cDNAs were generated from total RNA using MuLV reverse transcriptase (Applied Biosystems, Foster City, CA). Real-time quantitative RT-PCR analysis of hIL-2, HSV-TK, TG, thyroperoxidase (TPO), sodium/iodide symporter (NIS), TSH receptor (TSHR), and thyroid-specific transcription factor (TTF-1) was performed on the ABI Prism 7700 Sequence Detector (Applied Biosystems). Oligonucleotide primer sequences used to amplify hIL-2, HSV-TK (6), TPO, NIS, TSHR (12), and TTF-1 (13) have been reported elsewhere. Quantitative RT-PCR analysis was performed using SYBR Green PCR Core Reagents kit (Applied Biosystems), according to the manufacturer’s protocol. Quantitative RT-PCR analysis of the TG gene was performed using oligonucleotide primers and a fluorogenic Taqman probe (Applied Biosystems), as described (14). The hIL-2 and HSV-TK genes underwent absolute quantitation against a standard curve generated by amplification of the pMFGIL-2TKSN plasmid, whereas TG, TPO, TSHR, NIS, and TTF-1 underwent absolute quantitation against a standard curve obtained by amplification of correspondent cDNAs subcloned into the pCR2.1 vector (Invitrogen). Expression of all the target genes was also normalized to the endogenous control ß-actin mRNA, as quantitated by real-time RT-PCR analysis using TaqMan ß-actin RNA control reagent kit (Applied Biosystems).
In vitro cytotoxicity assay
Retrovirus-infected cells were seeded at density of 5 x 103 cells/well in 96-well microtiter plates. On the next day, the cells were treated with ganciclovir (GCV) (Sigma) concentrations ranging from 0.01 to 100 µM in 100-µl medium, either in the presence or absence of 10 IU/liter TSH. Cell survival was quantitated by the dimethylthiazoldiphenyltetrazoliumbromide (MTT) assay (10) 5 and 9 d later. Survival ratios were expressed as percentages relative to untreated controls.
In vivo antitumor effect
Male nude mice (Charles-River Italia Spa, Calco, Lecco, Italy), 6–7 wk of age, received sc injection on both flanks with 8 x 106 retrovirus-infected and parental cells in 150 µl PBS. After 7 d, ip injections of either 100 mg/kg GCV in PBS or PBS alone were performed daily for 1 wk. The perpendicular tumor diameters were measured using calipers, and tumor volumes were calculated by the formula of rotational ellipsoid: tumor volume = A x B2/2, where A is the longer diameter and B is the smaller diameter. The results were expressed as percentages relative to tumor size on d 0. None of the mice showed wasting or visible indications of toxicity. Animals were killed 24 h after the last GCV treatment, and tissues were harvested for pathological examination. All procedures were carried out following the guidelines recommended by Institutional Animal Care and Use Committee of the University of Padova.
Histology
Tissue specimens were fixed in buffered 4% formalin for 24 h and paraffin embedded after dehydration. Tissue slides measuring 5 µm were cut and stained with hematoxylin and eosin following routine histological methods. Microscopical examination was performed at low and high magnification, evaluating tumor size, necrosis, and inflammatory infiltration in a semiquantitative grade, as: -, absent/negative; +, moderate; ++, extensive.
Statistical analysis
Results are given as the mean ± SE. Comparisons between variables were tested by one-way ANOVA or Student’s t test, as appropriate. A P value less than 0.05 was considered statistically significant.
Results
Construction of thyroid-specific retroviral vectors and determination of retroviral vector titer
To generate transcriptionally targeted thyroid-specific retroviral vectors, the viral enhancer between the NheI and XbaI sites in the pMFG Moloney-derived vector was replaced with a 650-bp DNA fragment containing human TG enhancer (8) (Fig. 1
). Modifications were made in the 3' LTR so that they would be duplicated in the 5' LTR of the provirus. A similar transcriptional targeting approach by LTR reshuffling was first described by Vile et al. (3). The reporter vectors pMFGnlsLacZ(TGenh) and pMFGnlsLacZ were constructed by insertion of the nlsLacZ sequence, which encodes for an Escherichia coli ß-galactosidase preceded by a nuclear localization signal, into the vector backbone. Viral titer, as determined by infection of NIH3T3 cells with virus-containing supernatants from single cell-derived clones of stably transfected FLYA13 producer cells, ranged from 2 x 105 to 3 x 106 cf.u./ml for the MFGIL-2TKSN(TGenh) vector and from 2 x 105 to 3.5 x 106 cf.u./ml for the MFGIL-2TKSN vector (P = NS).
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To assess the feasibility of using the TG enhancer to target thyroid carcinoma cells, retroviral vectors containing the reporter gene nlsLacZ were used. Forty-eight hours after transient transfection of FLYA13 packaging cells, viral supernatant was used to infect target thyroid and nonthyroid cell lines. Expression of ß-galactosidase was evaluated by X-gal staining 48 h after infection. Viral titer, evaluated in infected NIH3T3 cells, was 400 cf.u./ml. The number of differentiated thyroid carcinoma cells (i.e. WRO, FTC-133, and C8305 cells) expressing ß-galactosidase after transduction either with the targeted vector or control vector was similar (P = NS), whereas in ARO anaplastic thyroid carcinoma cells and nonthyroid cells, ß-galactosidase expression was 10- to 100-fold lower after infection with the thyroid-specific vector MFGnlsLacZ(TGenh) than with the control vector MFGnlsLacZ (P < 0.05, Fig. 2). Although expression of ß-galactosidase was higher in TSH-treated differentiated thyroid cells transduced with the MFGnlsLacZ(TGenh) vector than the corresponding untreated cells, the difference was not statistically significant (data not shown).
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Expression of therapeutic genes in transduced cells was evaluated by quantitative real-time RT-PCR analysis. The levels of therapeutic gene expression in cells infected with the thyroid-specific vector MFGIL-2TKSN(TGenh) were significantly lower than those obtained after infection with the control vector MFGIL-2TKSN (Fig. 3). Expression of therapeutic genes was very low or undetectable in ARO anaplastic thyroid carcinoma cells and in nonthyroid cells (HeLa, HepG2, and AoU373) infected with the thyroid-targeted vector, whereas WRO, FTC-133, and C8305 differentiated thyroid carcinoma cells demonstrated significantly higher levels of transgene expression (Fig. 3). Treatment with TSH slightly increased therapeutic gene expression in differentiated thyroid carcinoma cells; however, the effect was not statistically significant (data not shown).
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To assess whether a relationship existed between transgene expression driven by the thyroid-specific vector and the differentiated phenotype of thyroid carcinoma cells, thyroid-specific mRNAs, including those for TG, TPO, NIS, TSHR, and TTF-1, were analyzed by real-time quantitative RT-PCR. Expression of thyroid-specific genes was high in WRO, FTC-133, and C8305 differentiated thyroid cells but very low or undetectable in ARO anaplastic carcinoma cells (Fig. 4).
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Morphological characteristics and proliferation rate of transduced cells remained the same as those of wild-type cells. Sensitivity to GCV of parental and infected thyroid and nonthyroid cells was assessed by determining the IC50 by the MTT assay. Data at 9 d of treatment with GCV are shown in Fig. 5. Infection with the MFGIL-2TKSN vector conferred sensitivity to GCV to all cell lines, with IC50 values ranging from 0.1–16 µM and from 0.04–3 µM, after 5 and 9 d of treatment, respectively. At variance, the efficacy of HSV-TK/GCV after infection with the MFGIL-2TKSN(TGenh) vector was dependent on the TG expression status of the different cell lines, being similar to the cytotoxic effect obtained after infection with the control vector in WRO, FTC-133, and C8305 cells. A 100- to 1000-fold lower cytotoxicity was observed in ARO anaplastic thyroid carcinoma cells and in nonthyroid HeLa, AoU373, and HepG2 cells transduced with the thyroid-specific vector. Treatment with 10 UI/liter TSH did not significantly influence GCV sensitivity of thyroid carcinoma cell lines.
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The in vivo efficacy of the retroviral vector constructs was evaluated in tumor models obtained by sc injection of infected and parental cells in nude mice. To investigate the in vivo effect of IL-2, the size of tumors obtained with transduced cells (i.e. expressing hIL-2) was compared with parental cell tumors. Mean volume of tumors obtained by injection of noninfected cells was larger than the volume of tumors of infected cells (mean volume after 7 d from cell injection, 75 ± 8.4 mm3 vs. 35 ± 5.2 mm3, respectively, P < 0.05). On histological examination, mice not receiving GCV treatment had large nodules of actively dividing cells without tumor necrosis or with small focal areas of necrosis and no significant inflammatory cell infiltration. Moderate infiltration of inflammatory cells was observed in infected tumors not receiving GCV treatment.
To investigate the in vivo efficacy of HSV-TK, mice were injected with tumor cells and, after 1 wk, treated with 100 mg/kg GCV daily for 1 wk. Treatment with GCV led to a complete or near complete (<20% of the original mass) regression of the volume of tumors infected with the MFGIL-2TKSN control vector. A significant reduction of tumor mass (0–18% of the original tumor volume) was observed also in differentiated thyroid cell tumors infected with the MFGIL-2TKSN(TGenh) vector. At variance, anaplastic thyroid carcinoma and nonthyroid tumors infected with the thyroid-specific vector showed a 2-fold increase of size after 1 wk of treatment with GCV (Fig. 6). Transduced cell tumors not treated with GCV showed 3- to 5-fold increase in size after 7 d. Histological analysis of tumor specimens from GCV-treated mice showed wide necrotic areas and a significant infiltration of inflammatory cells, with prevalence of neutrophils (Fig. 7).
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In the present study, we developed a transcriptionally targeted thyroid-specific retroviral vector by replacing the viral enhancer sequence with enhancer elements of the human TG gene, which is selectively expressed in thyroid tissue and differentiated thyroid carcinomas. Moreover, insertion of two therapeutic genes into the same vector backbone allowed a combined immunomodulating and suicide gene therapy approach.
Targeting therapeutic gene expression is an important issue in the development of gene therapy protocols for differentiated thyroid cancer. Indeed, thyroid cancer arises in the context of vital anatomic structures of the neck that should be spared by cytotoxic genes. At variance, there is no need to spare residual normal thyroid tissue, which is generally completely removed to allow early detection of relapsing tumor by monitoring blood TG levels. Enhancer/promoter sequences of thyroid-specific genes are good candidates to drive expression of therapeutic genes. Moreover, the use of TSH-responsive transcriptional control elements, such as the TG enhancer/promoter, would allow increasing transgene expression by TSH treatment. This aspect has important clinical implications following introduction of recombinant human TSH in the management of patients operated for differentiated thyroid cancer.
The feasibility of transcriptionally targeting thyroid cancer cells has already been demonstrated with retroviral and adenoviral vectors expressing HSV-TK under the control of TG promoter (15, 16). A major limitation of this approach, however, was the low efficiency of the cellular promoter, compared with the strong viral promoters. Enhancement of TG promoter activity and specificity was obtained by introducing a TG enhancer upstream the TG promoter (17). Another strategy to improve thyroid-specific expression of therapeutic genes was the use of a Cre-loxP system, in which the Cre recombinase was expressed when the TG promoter was active, thus switching on HSV-TK expression (18).
Replacement of viral enhancer in retroviral LTR with corresponding cellular sequences, although maintaining viral promoter, can sustain high-level, tissue-specific expression with high viral titers (4). This strategy obviates the problem of transcriptional interference because of the presence of a functional LTR in the integrated provirus. We followed a similar approach to construct a thyroid-specific retroviral vector. Our results show that the ß-galactosidase reporter gene and the therapeutic genes under control of the chimeric LTR were selectively expressed in target differentiated thyroid carcinoma cells, with very low or absent expression in anaplastic thyroid carcinoma and nonthyroid cells. These results confirm the observations by Berg et al. (8), who demonstrated the ability of human TG enhancer to induce thyroid-specific transgene expression. In our study, selective thyroid-specific transgene expression paralleled expression of thyroid-specific genes, being higher in TG- and TTF-1-expressing cells and very low or absent in anaplastic thyroid carcinoma cells and nonthyroid cells, which express very low or absent amounts of TG. Interestingly, reexpression of TTF-1 in TG-negative anaplastic thyroid carcinoma cells induced TG expression and conferred sensitivity to transcriptionally targeted therapeutic genes under control of the TG promoter (19). In our study, TSH treatment induced only a slight increase of transgene expression in transduced thyroid cells, although not significant. However, it should be borne in mind that thyroid cell lines accumulate a number of genetic abnormalities with impairment of the TSH signal transduction pathway and do not exactly reproduce the clinical phenotype of differentiated thyroid carcinomas, which generally show thyroid differentiated functions, including TSH responsiveness (20). Thus, if this therapeutic approach is used in patients with differentiated thyroid cancer, it is conceivable that treatment with TSH would significantly increase therapeutic gene expression driven by the targeted vector, besides stimulating cell proliferation and thus GCV incorporation into tumor cells.
Low efficiency of cellular enhancer/promoter sequences is a commonly encountered problem in the design of targeted vectors. Also in our experience, human TG enhancer was weaker than viral enhancer, even if it showed good tissue specificity, as demonstrated by quantitative RT-PCR analysis of transgene expression. However, in vitro and in vivo experiments showed a comparable cytotoxic effect of GCV in differentiated thyroid carcinoma cells transduced with the targeted vector or with the control vector. It is therefore conceivable that even relatively low levels of HSV-TK gene product are enough to kill GCV-treated cells.
Another possible drawback of a transcriptionally targeted vector obtained by LTR reshuffling is the decrease in titer, compared with vectors with wild-type LTR. In our experience, titers of the retroviral vector with chimeric LTR were similar to those of the control vector, having a wild-type LTR. Thus, the introduction of a heterologous and larger DNA sequence (650 bp of the TG enhancer vs. 267 bp of viral enhancer) in the U3 region did not significantly influence viral titer. These results confirm the observation that titer reduction of vectors with chimeric LTR occurs when reshuffling involves the R region of LTR (4).
To amplify the antitumor efficacy of gene therapy, we designed a new strategy by constructing a bicistronic retroviral vector that coexpresses a suicide gene (HSV-TK) along with a cytokine gene (hIL-2) (5). This combined therapeutic approach, we successfully used in a pilot study in patients with recurrent glioblastoma multiforme (6), was also pursued in the present study of gene therapy for thyroid cancer. The superiority of combined immunomodulating and suicide gene therapy for cancer over strategies based on a single therapeutic gene has been well documented in several studies in animal models of cancer (21, 22, 23), including medullary thyroid carcinoma (24, 25). Our in vivo studies in nude mice injected with transduced tumor cells demonstrated a nonspecific antitumor immune response generated by IL-2 gene expression. In particular, transduced tumor cells showed growth retardation, compared with parental nontransduced cells, associated with focal necrosis and neutrophil infiltrates at histology. This antitumor effect was dramatically enhanced by treatment with GCV, which induced a near complete or complete regression of cell tumors transduced with the nontargeted vector. At variance, infection with the thyroid-targeted vector led to regression of only TG-expressing tumors, but TG-negative tumors showed a 3- to 5-fold increase in size. Thus, retroviral vectors, although intrinsically not suited for systemic delivery (26), confirm the lack of side effects when also expressing a combination of different therapeutic genes.
In conclusion, our results indicate that replacement of the viral enhancer with the TG enhancer confers selectivity of transgene expression in thyroid cells. Thus, the combined thyroid-specific expression of two therapeutic genes (cytokine and suicide genes), although representing a safe tumor-targeted treatment, allows for an increased anticancer effect. The efficacy of this therapeutic approach is being evaluated in our ongoing pilot study in humans with advanced thyroid cancer treated by intratumoral injection of retroviral vector producing cells.
Acknowledgments
Footnotes
This work was supported by a MURST Grant (2001061979), National Research Council Grants (99-02549-CT04 and 01-00777-PF49, to G.P.), and a grant from Regione Veneto (R.S.F. 966/02/00, to M.B.).
L.B. and R.B. equally contributed to the present work.
Abbreviations: FBS, Fetal bovine serum; FTC, follicular thyroid carcinoma; GCV, ganciclovir; hIL-2, human IL-2; HSV-TK, herpes simplex virus thymidine kinase; LTR, long-terminal repeat; MTT, dimethylthiazoldiphenyltetrazoliumbromide; NIS, sodium/iodide symporter; SV40, Simian virus 40; TG, thyroglobulin; TPO, thyroperoxidase; TSHR, TSH receptor; TTF-1, thyroid-specific transcription factor 1.
Received June 25, 2002.
Accepted August 14, 2002.
References
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