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Endocrinology, doi:10.1210/en.2002-0206
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Endocrinology Vol. 144, No. 8 3423-3432
Copyright © 2003 by The Endocrine Society

Retinoic Acid-Induced Stimulation of Sodium Iodide Symporter Expression and Cytotoxicity of Radioiodine in Prostate Cancer Cells

C. Spitzweg, I. V. Scholz, E. R. Bergert, D. J. Tindall, C. Y. F. Young, B. Göke and J. C. Morris

Department of Internal Medicine II (C.S., I.V.S., B.G.), Klinikum Grosshadern, Ludwig-Maximilians-University, Munich 81377, Germany; and Departments of Endocrinology (E.R.B., J.C.M.) and Urology (D.J.T., C.Y.F.Y.), Mayo Clinic, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Christine Spitzweg, M.D., Klinikum Grosshadern, Medizinische Klinik II, Marchioninistrasse 15, 81377 Muenchen, Germany. E-mail: spitzweg.christine{at}gmx.net.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We reported recently the induction of androgen-dependent iodide uptake activity in the human prostatic adenocarcinoma cell line LNCaP using a prostate-specific antigen (PSA) promoter-directed expression of the sodium iodide symporter (NIS) gene. This offers the potential to treat prostate cancer with radioiodine. In the current study, we examined the regulation of PSA promoter-directed NIS expression and therapeutic effectiveness of 131I in LNCaP cells by all-trans-retinoic acid (atRA). For this purpose, NIS mRNA and protein expression levels in the NIS-transfected LNCaP cell line NP-1 were examined by Northern and Western blot analysis following incubation with atRA (10 -9 to 10-6 M) in the presence of 10-9 M mibolerone (mib). In addition, NIS functional activity was measured by iodide uptake assay, and in vitro cytotoxicity of 131I was examined by in vitro clonogenic assay. Following incubation with atRA, NIS mRNA levels in NP-1 cells were stimulated 3-fold in a concentration-dependent manner, whereas NIS protein levels increased 2.3-fold and iodide accumulation was stimulated 1.45-fold. This stimulatory effect of atRA, which has been shown to be retinoic acid receptor mediated, was completely blocked by the pure androgen receptor antagonist casodex (10-6 M), indicating that it is androgen receptor dependent. The selective killing effect of 131I in NP-1 cells was 50% in NP-1 cells incubated with 10-9 M mib. This was increased to 90% in NP-1 cells treated with atRA (10-7 M) plus 10-9 M mib. In conclusion, treatment with atRA increases NIS expression levels and selective killing effect of 131I in prostate cancer cells stably expressing NIS under the control of the PSA promoter. Therefore atRA may be used to enhance the therapeutic response to radioiodine in prostate cancer cells following PSA promoter-directed NIS gene delivery.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE UNIQUE PROPERTY of thyroid follicular cells to trap and concentrate iodide because of expression of the sodium iodide symporter (NIS) allows imaging as well as effective therapy of differentiated thyroid carcinomas and their metastases by administration of radioiodine. Thus, the prognosis and treatment of thyroid cancer is improved significantly (1). Cloning of the human and rat NIS genes in 1996 and their extensive characterization since then have paved the way for the development of a novel cytoreductive gene therapy strategy for the treatment of extrathyroidal malignancies based on targeted NIS gene transfer followed by radioiodine therapy (2, 3, 4, 5). NIS gene transfer using tissue- specific promoters provides a way of selectively targeting the NIS gene to tumor cells, thereby maximizing tissue-specific cytotoxicity with minimal toxic side effects in other organs (6).

In contrast to thyroid cancer, despite a variety of treatment options including androgen ablation, surgery, external radiotherapy, interstitial brachytherapy, and systemic cytotoxic chemotherapy, no curative therapy for metastatic prostate cancer exists, which still represents the second leading cause of cancer death in men (7). Therefore, the development and evaluation of novel treatment strategies, including prostate-specific antigen (PSA) promoter-targeted NIS gene transfer followed by radioiodine therapy, are urgently needed. Gene therapeutic approaches for the treatment of prostate cancer are attractive because of the possibility of selective targeting of therapeutic genes to tumor cells by application of tissue-specific promoters, thereby reducing extratumoral toxicities associated with treatments such as cytotoxic chemotherapy. We therefore chose prostate cancer as a tumor model to investigate the feasibility of radioiodine therapy of nonthyroidal tumors following NIS gene transfer.

The PSA gene is regulated strictly in a tissue-specific manner. Thus, the PSA promoter represents an important tool for prostate cell-specific gene delivery (8, 9, 10). A 6-kb PSA promoter fragment (11) has therefore been used in our studies to induce tissue-specific androgen-dependent iodide uptake activity in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP in vitro by liposome-mediated NIS gene delivery (12). LNCaP cells stably transfected with the human NIS gene under the control of the PSA promoter revealed androgen-dependent and prostate-specific iodide uptake activity that was high enough to allow a therapeutic effect of accumulated 131I in an in vitro clonogenic assay and in vivo in xenografts in nude mice (12, 13, 14). These studies clearly showed the potential of NIS as a novel therapeutic gene allowing radioiodine therapy of nonthyroidal cancers, in particular prostate cancer.

Retinoids, synthetic and natural analogs of vitamin A, play a well-characterized role in cancer development, cell differentiation, and cell growth (15). Induction of NIS expression and radioiodine uptake has been demonstrated in breast cancer cells following treatment with all-trans-retinoic acid (atRA), allowing a therapeutic effect of 131I in vitro (16). In addition, atRA has been shown to suppress iodide uptake and NIS mRNA levels in normal, nontransformed FRTL-5 cells, whereas NIS mRNA expression levels were up-regulated in human follicular thyroid carcinoma cell lines in vitro (17). A clinical study in 20 patients with advanced thyroid cancer (8 follicular, 7 papillary, 5 oxyphilic) showed that 13-cis-retinoic acid treatment (1.5 mg/kg·d for 5 wk) was capable of reinducing iodine uptake in 50% of tumors (18). Treatment with retinoic acid may therefore provide a means of reestablishing the therapeutic efficacy of radioiodine therapy by targeted up-regulation of iodide transport in thyroid cancer cells and down-regulating iodide accumulation in surrounding normal thyroid tissue. Furthermore, atRA has been reported to inhibit cell cycle progression and induce apoptosis in many tumor cell lines. Also, it has been used in a number of clinical studies to investigate the therapeutic effect of retinoids in a variety of tumors, including prostate cancer (15, 19, 20). Retinoids have been reported to increase PSA secretion in LNCaP cells and inhibit their cell growth by an androgen receptor-dependent mechanism preceded by the stimulation of androgen receptor gene expression (21, 22, 23).

In the current study, we therefore used atRA to examine the effect of retinoids on androgen-dependent NIS expression and therapeutic response to 131I in LNCaP cells stably expressing the human NIS gene under the control of the PSA promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid constructs and NIS gene transfer
The expression and control vectors have been generated as previously described (12). We used the pEGFP-1 vector (CLONTECH Laboratories, Inc., San Diego, CA) that had been precut with HindIII and NotI restriction enzymes, thereby removing the 800-bp EGFP fragment. The resulting expression plasmid construct contained full-length human NIS cDNA coupled to the 6-kb PSA promoter fragment (NIS/PSA-pEGFP-1). Two control vectors were designed containing NIS cDNA without the PSA promoter (NIS-pEGFP-1) and the PSA promoter without NIS cDNA (PSA-pEGFP-1).

Transient and stable transfection of prostate cancer cells was performed as previously described (12). In brief, the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP and androgen-independent prostate cancer control cell lines without PSA expression (PC-3, DU-145) were transfected with NIS/PSA-pEGFP-1 and the control vectors NIS-pEGFP-1 and PSA-pEGFP-1, respectively, using LipofectAMINE Plus Reagent (Life Technologies, Inc., Gaithersburg, MD). Selection for stable transfection of LNCaP cells was performed with geneticin and surviving clones were isolated and subjected to screening for androgen-dependent iodide uptake activity. NP-1, the LNCaP cell clone with the highest androgen-dependent iodide uptake activity was chosen for the studies as well as the stably transfected LNCaP control cell lines P-1 (PSA-pEGFP-1) and N-1 (NIS-pEGFP-1).

Cell culture and treatments
LNCaP, PC-3, and DU-145 cells were grown in RPMI 1640 medium (Life Technologies, Inc., Karlsruhe, Germany) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) at 37 C and 5% CO2. Following incubation in serum-free RPMI 1640 medium for 24 h or transient transfection, respectively, cells were incubated with mibolerone (mib; synthetic androgen, which is not metabolized by LNCaP cells and has similar affinity to the androgen receptor as dihydrotestosterone) (10-11 to 10-7 M), atRA (10-9 to 10-6 M) (Sigma, Taufkirchen, Germany), 9-cis-retinoic acid (9-cis-RA), TTNPB (4-[E2–5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl]) (Sigma), and the antiandrogen casodex (bicalutamide, 10-6 M, a generous gift from AstraZeneca, Macclesfield, UK) in the presence of 10% charcoal-stripped fetal bovine serum for 24–72 h.

Cell proliferation assay
Cell proliferation was measured using the commercially available MTS-assay (Promega Corp., Mannheim, Germany) according to the manufacturer’s recommendations. Cells were incubated with freshly prepared MTS [3-(4,5-dimethylthiazol)-2-yl-5-(3-carboxymeth-oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)]/phenazine methosulfate solution (ratio 1:1 by volume) for 1.5 h at 37 C in a humidified 5% CO2 atmosphere. The absorbance of the formazan product was read at 490 nm, which is directly proportional to the number of living cells in culture.

Iodide uptake studies
Uptake of 125I by untreated and treated prostate cancer cells was determined at steady-state conditions as described by Weiss et al. (24). In brief, cells were plated on 6-well plates (2 x 105 cells/well), and following incubation with mib (10-11 to 10-7 M) and atRA (10-9 to 10-6 M), respectively, iodide uptake studies were performed in Hanks’ balanced salt solution (HBSS) supplemented with 10 µM NaI, 0.1 µCi Na 125I/ml, and 10 mM HEPES (pH 7.3). A 100-µM concentration of KClO4 was added to control wells. Trapped iodide was removed from cells by a 20-min incubation in 1 N NaOH and measured by {gamma}-counting. Results were normalized to cell survival measured by cell proliferation assay (see above) and expressed as cpm/A490 nm.

Measurement of iodide organification
Cells, grown in 6-well plates, were incubated for 2 h at 37 C with HBSS supplemented with 10 µM NaI, 0.1 µCi Na 125I/ml, and 10 mM HEPES (pH 7.3). Contents of organified iodide in the cells were determined by trichloroacetic acid precipitation as described by Urabe et al. (25).

Iodide efflux studies
Efflux of 125I was determined as described by Weiss et al. (24). In brief, cells were plated on 6-well plates (2 x 105 cells/well), following incubation with mib (10-9 M) and atRA (10-7 M), respectively, cells were incubated with HBSS supplemented with 10 µM NaI, 0.1 µCi Na 125I/ml and 10 mM HEPES (pH 7.3) at 37 C for 1 h. Medium was then replaced every 5 min with fresh HBSS. The content of 125I in the collected supernatant was measured by {gamma}-counter. After the last time point, trapped 125I was removed from cells by a 20-min incubation in 1 N NaOH and measured by {gamma}-counting.

Membrane preparation
Cell membranes were prepared from treated and untreated LNCaP cells by a modification of a previously described procedure (26). In brief, cells plated on 100-mm dishes were washed with PBS, harvested, and resuspended in buffer A, consisting of 250 mM sucrose, 10 mM HEPES (pH 7.5), 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethanesulfonyl fluoride. The homogenate was centrifuged twice at 500 x g for 15 min at 4 C. Following centrifugations, 100 µl 1 M Na2CO3 /ml buffer A was added to the supernatant and incubated at 4 C for 45 min with continuous shaking. Then a further centrifugation at 100,000 x g was performed for 15 min, and the pellet was resuspended in an appropriate volume of buffer B consisting of 250 mM sucrose, 10 mM HEPES (pH 7.5), and 1 mM MgCl2. Protein concentrations were determined by a protein assay (DC protein assay, Bio-Rad Laboratories, Inc., Munich, Germany).

Western blot analysis
For Western blot analysis, the NuPAGE electrophoresis system (Invitrogen, Karlsruhe, Germany) was used. Equal amounts of membrane protein as determined by DC protein assay (10 µg) (Bio-Rad Laboratories, Inc.) were reduced by incubation with 0.5 M dithiothreitol for 10 min at 70 C and loaded on 4–12% Bis-Tris-HCl buffered polyacrylamide gels. Following gel electrophoresis for 1 h, proteins were transferred to nitrocellulose membranes using electroblotting, and transfer of equal amounts of membrane protein was confirmed by colloidal gold staining (Bio-Rad Laboratories, Inc.). Following blotting, membranes were preincubated for 1 h in 5% low-fat dried milk in TBS-T (20 mM Tris, 137 mM NaCl, and 0.1% Tween-20) to block nonspecific binding sites. Membranes were then incubated with a mouse monoclonal antibody directed against amino acid residues 468–643 of human NIS (dilution 1:3000) (27) for 2 h at room temperature. After washing with TBS-T, horseradish peroxidase-labeled goat-antimouse antibody was applied (dilution 1:5000) for 1 h at room temperature before incubation with enhanced chemiluminescence Western blotting detection reagents (Amersham, Braunschweig, Germany) for 1 min. Exposures were made at room temperature for approximately 1 min using BIOMAX MR films (Kodak, Rochester, NY). Prestained protein molecular weight standards (Life Technologies, Inc.) were run in the same gels for comparison of molecular weight and estimation of transfer efficiency. For quantitative analysis computer-assisted densitometric analysis of band intensities was performed.

RNA preparation and Northern blot analysis
Total RNA was isolated from untreated and treated LNCaP cells, respectively, by the modified acid guanidinium thiocyanate-phenol-chloroform method according to Chomczynski and Sacchi (28) using the Rneasy Mini kit (QIAGEN, Hilden, Germany). Aliquots (15 µg) of RNA were electrophoresed on a 1% agarose gel containing 2 M formaldehyde and transferred overnight in 20 x saline sodium citrate (SSC) to a positively charged nylon membrane (QIAGEN). The human NIS-gene-specific cDNA-fragment (nucleotides 1184–1667), which was generated as described previously (29), was radiolabeled with [{alpha}-32P]dATP by random priming (Amersham), and used as a hybridization probe. Blots were prehybridized at 68 C in hybridization solution (Express Hyb solution, CLONTECH Laboratories, Inc., Heidelberg, Germany) for 30 min, followed by hybridization at 68 C for 1 h. Blots were then rinsed four times in 2 x SSC/0.05% SDS at room temperature for 10 min and twice in 0.1 x SSC/0.1% SDS at 50 C for 20 min, respectively. Exposures were made at -80 C for 48 h using X-OMAT AR films (Kodak). To strip off the NIS cDNA probe, blots were treated in 0.5% SDS at 95 C for 10 min and reprobed with a rat ß-actin cDNA probe to monitor RNA integrity and quantity. Computer-assisted densitometric analysis of band intensities was performed, and NIS measurements were normalized for ß-actin signal intensity.

In vitro clonogenic assay
The in vitro clonogenic assay was performed as described by Mandell et al. (30). In brief, untreated and treated LNCaP cells were grown to 50% confluency, washed with HBSS, and incubated for 7 h with 10 ml of 80 µCi/ml Na131I in HBSS supplemented with 10 µM NaI and 10 mM HEPES at pH 7.3. Following incubation with radioiodine, cells were washed with HBSS, trypsinized using 0.05% trypsin-EDTA, counted, and plated in quadruplicates at cell densities of 1000, 2000, 3000, 5000, and 7000 cells/well in 12-well plates. Four weeks later, after colony development, cells were fixed with methanol, stained with crystal violet, and colonies containing more than 50 cells were counted. Parallel experiments were performed for each cell line using HBSS without 131I, and all values were adjusted for plating efficiency. The percentage of survival represents the percentage of cell colonies after 131I treatment, compared with mock treatment with HBSS. Results are expressed as means ± SEM of quadruplicates. Statistical significance was tested using t test.

Statistical methods
All experiments were carried out in triplicates. Results are presented as mean ± SEM of triplicates. Statistical significance was tested using t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Iodide uptake studies in vitro
The effect of atRA on iodide accumulation was examined in LNCaP cells following PSA promoter-directed NIS gene transfer (NP-1) as well as in control cell lines P-1, N-1, and in androgen-independent control cell lines PC-3 and DU-145. For this purpose cells were incubated with or without mib (10-9 M) and atRA (10-9 to 10-6 M), respectively, and iodide accumulation was measured using an iodide uptake assay (Fig. 1Go, A–E). A 48-h treatment with atRA (10-9 to 10-7 M) increased androgen-dependent and perchlorate-sensitive iodide accumulation in NP-1 cells approximately up to 1.45-fold in a concentration-dependent and time-dependent manner. Maximal stimulation of iodide accumulation was seen after 48 h at 10-7 M atRA (Fig. 1Go, A and C). The iodide uptake following incubation with mib and atRA was completely blocked by the pure androgen receptor antagonist casodex (10-6 M), indicating that the effect of atRA is androgen receptor dependent (Fig. 1AGo). No iodide accumulation above background level was observed in androgen-deprived NP-1 cells when incubated in the presence or in the absence of atRA (10-9 to 10-6 M) (Fig. 1AGo). Iodide accumulation in NP-1 cells was stimulated by mib (10-11 to 10-7 M) in a concentration-dependent manner with maximal stimulation at 10-8 M mib (Fig. 1BGo). To evaluate whether the stimulation of iodide uptake is retinoic acid receptor (RAR) mediated, we examined the effect of several retinoid compounds on iodide accumulation in NP-1 cells (Fig. 1DGo). The RAR-specific ligand TTNPB increased iodide uptake in NP-1 cells in the presence of mib (10-9 M) to the same level as atRA, suggesting that RAR mediates the effect. 9-cis-RA, which activates RAR as well as retinoid X receptor, also had a similar effect as atRA on iodide accumulation in NP-1 cells. Furthermore, in contrast to NP-1 cells, no perchlorate-sensitive iodide uptake was observed in P-1 and N-1 cells incubated in the presence or absence of mib or atRA, respectively (Fig. 1EGo). No iodide accumulation was detected in control cell lines PC-3 and DU-145 following transient transfection with NIS/PSA-pEGFP-1 in the presence or absence of mib and atRA, respectively (Fig. 1EGo). Taken together, treatment with atRA significantly increased androgen-induced iodide accumulation in NP-1 cells in a concentration- and time-dependent manner. This effect has been shown to be RAR mediated and androgen receptor dependent.



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  		FIG. 1. A, Effect of atRA on iodide accumulation in LNCaP cells following PSA promoter-directed NIS gene transfer. LNCaP cells stably expressing NIS under the control of the PSA promoter (NP-1) were incubated with or without mib (10-9 M) and atRA (10-9 to 10-6 M), respectively, and iodide accumulation was measured as described by Weiss et al. (24 ). A 48-h incubation with atRA in the presence of mib increased androgen-dependent and perchlorate-sensitive iodide accumulation in NP-1 cells up to 1.45-fold in a concentration-dependent manner. No perchlorate-sensitive iodide uptake was observed in androgen-deprived cells in the presence or absence of atRA. The iodide uptake following incubation with mib and atRA was completely blocked by the androgen receptor antagonist casodex. Results have been normalized to cell proliferation measured by cell proliferation assay and are expressed as percent of control. Values are mean ± SEM of triplicate experiments. *, P < 0.01. B, Concentration-dependent stimulation of iodide uptake in LNCaP cells following PSA promoter-directed NIS gene transfer by androgen. Iodide accumulation in NP-1 cells was assayed as described by Weiss et al. (24 ) following incubation with mib (10-11 to 10-7 M). Iodide accumulation was stimulated in a concentration-dependent manner with maximal stimulation at 10-8 M mib. Results have been normalized to cell proliferation measured by cell proliferation assay and expressed as cpm/A490 nm. Values are mean ± SEM of triplicate experiments. *, P < 0.05; **, P < 0.01. C, Time-dependent stimulation of iodide uptake in LNCaP cells following PSA promoter-directed NIS gene transfer by atRA. Iodide accumulation in NP-1 cells was assayed as described by Weiss et al. (24 ) following incubation with atRA (10-7 M) in the presence of mib (10-9 M). Iodide accumulation was stimulated in a time-dependent manner with maximal stimulation at 48 h. Results have been normalized to cell proliferation measured by cell proliferation assay and expressed as cpm/A490 nm. Values are mean ± SEM of triplicate experiments. *, P < 0.05; **, P < 0.01. D, Effect of several retinoid compounds on iodide uptake in LNCaP cells following PSA promoter-directed NIS gene transfer. Iodide accumulation in NP-1 cells was assayed as described by Weiss et al. (24 ) following incubation with mib (10-9 M) alone and in the presence of atRA (10-7 M), 9-cis-RA (10-7 M), and TTNPB (10-7 M). 9-cis-RA as well as TTNPB had similar effects on iodide accumulation in NP-1 cells as atRA. Results have been normalized to cell proliferation measured by cell proliferation assay and expressed as percent of control. Values are mean ± SEM of triplicate experiments. E, Effect of atRA on iodide accumulation in control cell lines. Transiently transfected androgen-independent prostate cancer control cell lines PC-3 and DU-145 (NIS/PSA-pEGFP-1), stably transfected LNCaP control cell lines P-1 (PSA-pEGFP-1) and N-1 (NIS-pEGFP-1), and NP-1 cells (NIS/PSA-pEGFP-1) were incubated with or without mib (10-9 M) and atRA (10-7 M), respectively, and iodide accumulation was measured as described by Weiss et al. (24 ). In contrast to NP-1 cells, which showed an up to 1.45-fold increase of androgen-dependent and perchlorate-sensitive iodide accumulation after incubation with atRA in the presence of mib, no iodide accumulation was detected in PC-3, DU-145, P-1, and N-1 cells in the presence or absence of mib and atRA, respectively. Results have been normalized to cell proliferation measured by cell proliferation assay and expressed as cpm/A490 nm. Values are mean ± SEM of triplicate experiments.

 
Measurement of iodide organification
To investigate whether trapped iodide is organified in LNCaP cells following PSA promoter-directed NIS gene transfer (NP-1) and determine the effect of atRA on iodide organification, cells were incubated with or without atRA (10-7 M) in the presence of mib (10-9 M) and iodide organification was measured by trichloroacetic acid precipitation of cell lysate after the incubation of the cells with 0.1 µCi Na125I/ml (Fig. 2AGo). No organification was observed in NP-1 cells incubated with mib alone, which was not changed by additional treatment with atRA.



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  		FIG. 2. A, Effect of atRA on iodide organification. LNCaP cells following PSA promoter-directed NIS gene transfer (NP-1) were incubated with mib (10-9 M) in the presence or absence of atRA (10-7 M) for 48 h, and iodide organification was measured by trichloroacetic acid precipitation as described by Urabe et al. (25 ). Nonspecific binding of 125I was determined in the presence of KClO4. No organification of iodide was observed in NP-1 cells incubated with mib alone, which was not changed by additional treatment with atRA. B, Effect of atRA on iodide efflux. LNCaP cells following PSA promoter-directed NIS gene transfer (NP-1) were incubated with mib (10-9 M) in the presence or absence of atRA (10-7 M) for 48 h, and iodide efflux was measured as described by Weiss et al. (24 ). Iodide efflux was rapid in NP-1 cells, more than 70% of the accumulated 125I was released into the medium during the initial 5 min, and treatment with atRA had no significant effect on the kinetics of iodide efflux.

 
Iodide efflux studies
To determine the effect of atRA on iodide efflux in LNCaP cells following PSA promoter-directed NIS gene transfer (NP-1), cells were incubated with or without atRA (10-7 M) in the presence of mib (10-9 M) and an iodide efflux assay was performed (Fig. 2BGo). Iodide efflux was rapid in NP-1 cells, more than 70% of the accumulated 125I was released into the medium during the initial 5 min, and treatment with atRA had no effect on the kinetics of iodide efflux.

Northern blot analysis
The effect of atRA on NIS mRNA expression was examined in LNCaP cells following PSA promoter-directed NIS gene transfer (NP-1). For this purpose cells were incubated with or without mib (10-9 M) and atRA (10-9 to 10-6 M), respectively, and NIS RNA steady-state levels were examined by high-stringency Northern blot analysis using a 32-P-labeled human NIS-specific cDNA probe (Fig. 3Go). Following a 48-h incubation with atRA (10-9 to 10-6 M) in the presence or absence of mib (10-9 M), NIS mRNA was detected as a single species of approximately 4 kb (Fig. 3Go, top). When normalized for ß-actin mRNA signal intensities (Fig. 3Go, middle), treatment with atRA (10-9 to 10-7 M) increased NIS mRNA levels up to 3-fold in a concentration-dependent manner (Fig. 3Go, bottom). No NIS mRNA expression was detected in androgen-deprived NP-1 cells when incubated in the presence (data not shown) or absence of atRA (10-9 to 10-6 M).



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  		FIG. 3. Effect of atRA on NIS mRNA. Northern blot analysis of mRNA derived from untreated NP-1 cells and NP-1 cells treated for 48 h with atRA (10-9 to 10-6 M) in the presence of mib (10-9 M) using a 32-P-labeled human NIS-specific cDNA probe. NIS mRNA was detected as a single species of approximately 4 kb (top). NIS mRNA levels were increased up to 3-fold in a concentration-dependent manner following incubation with atRA (10-9 to 10-7 M). Hybridization with ß-actin probe served as a control (middle). Computer-assisted densitometric analysis of band intensities was performed, and NIS measurements were normalized for ß-actin signal intensity (bottom). *, P < 0.01; **, P < 0.001.

 
Western blot analysis
The effect of atRA on NIS protein expression was examined in LNCaP cells following PSA promoter-directed NIS gene transfer (NP-1). For this purpose cells were incubated with or without mib (10-9 M) and atRA (10-9 to 10-6 M), respectively, and NIS protein expression levels were examined by Western blot analysis using a mouse monoclonal human NIS-specific antibody. NIS protein was detected as a band of approximately 90 kDa (Fig. 4Go, top). Treatment with atRA (10-9 to 10-7 M) increased NIS protein levels up to 2.3-fold in a concentration-dependent manner (Fig. 4Go, bottom). No NIS protein expression was detected in androgen-deprived NP-1 cells when incubated in the presence (data not shown) or absence of atRA (10-9 to 10-6 M).



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  		FIG. 4. Effect of atRA on NIS protein. Western blot analysis of untreated NP-1 cells and NP-1 cells treated with atRA using a mouse monoclonal human NIS-specific antibody. NIS protein was detected as a band of approximately 90 kDa (top). Following a 48-h treatment with atRA (10-9 to 10-7 M) in the presence of mib (10-9 M), NIS protein expression was increased up to 2.3-fold in a concentration-dependent manner. Computer-assisted densitometric analysis of band intensities was performed (bottom). *, P < 0.05; **, P < 0.01.

 
In vitro clonogenic assay
To determine the effect of atRA treatment on cytotoxicity of 131I in vitro in NP-1 and P-1 cells incubated in the presence or in the absence of androgen a clonogenic assay was performed (Fig. 5Go). Whereas only about 15% of P-1 cells were killed by exposure to 131I, approximately 50% of NP-1 cells incubated for 48 h with 10-9 M mib were killed in an in vitro clonogenic assay. Following treatment with 10-7 M atRA for 48 h in the presence of 10-9 M mib, approximately 90% of NP-1 cells were selectively killed by 131I. In androgen-deprived NP-1 cells incubated in the presence or absence of atRA, the cytotoxic effect of 131I was similar to the one seen in P-1 cells (data not shown). No cytotoxic effect of atRA (10-7 M) was detected in NP-1 cells incubated in the presence or absence of androgens without 131I.



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  		FIG. 5. Effect of atRA on in vitro cytotoxicity of 131-I. LNCaP cells following PSA promoter-directed NIS gene transfer (NP-1) and control cells stably transfected with the control vector PSA-/pEGFP-1 (P-1) were treated with or without atRA (10-7 M) in the presence of mib (10-9 M) for 48 h and then incubated with 0.8 mCi 131I for 7 h. Cells were then harvested and applied to clonogenic assay as described (30 ). Although only about 15% of P-1 cells were killed by exposure to 131I, approximately 50% of NP-1 incubated for 48 h with mib were killed in an in vitro clonogenic assay. Following treatment with atRA in the presence of mib, approximately 90% of NP-1 cells were selectively killed by 131I. Results are expressed as means ± SEM of quadruplicate experiments. *, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings in the present study demonstrate the effect of retinoids on androgen-dependent expression of the NIS and therapeutic response to 131I in LNCaP cells stably expressing the human NIS gene under the control of the PSA promoter. NIS mRNA and protein levels as well as iodide accumulating activity were stimulated following treatment with atRA plus androgen. This resulted in a significant increase of the efficacy of radioiodine therapy in prostate cancer cells following PSA promoter-directed NIS gene transfer.

NIS is an intrinsic membrane protein that mediates the active iodide uptake across the basolateral membrane of benign and malignant thyroidal cells (2, 3, 4, 31). Diagnostic imaging and effective therapy of differentiated thyroid carcinomas and their metastases by administration of radioiodine are based on thyroidal expression of functionally active NIS protein. Since cloning of the NIS gene in 1996, several investigators explored the capacity of NIS gene transfer into nonthyroidal tumor cells to induce radioiodine accumulation, thereby allowing radioiodine therapy of extrathyroidal malignancies. Using different gene delivery techniques, including electroporation, liposomes, and adenoviral and retroviral vectors, radioiodine accumulation was induced in vitro and in vivo in a variety of cancer cell lines (dedifferentiated thyroid cancer cells; glioma and neuroblastoma cells; melanoma, cervix, breast, lung, liver, colon, and ovarian carcinoma cells) by NIS gene delivery (30, 32, 33, 34, 35, 36, 37, 38, 39, 40). In an earlier study, we explored the efficacy of NIS gene transfer to induce accumulation as well as a therapeutic effect of 131I in stably transfected prostate cancer cells (12, 13) using the PSA promoter to target NIS gene expression to prostate cells, thereby minimizing extratumoral cytotoxicity (6, 8, 9, 11). Prostate cell-specific, androgen-regulated iodide uptake activity was demonstrated in LNCaP cell lines stably expressing NIS under the control of the PSA promoter in vitro and in vivo. Furthermore, the amount of accumulated 131I was shown to be sufficiently high to selectively kill NIS-transfected LNCaP cells in vitro as well as in vivo in LNCaP cell xenografts (12, 13, 14). These studies clearly showed for the first time that tissue-specific NIS gene delivery into nonthyroidal tumors is capable of inducing the accumulation of therapeutically effective radioiodine doses in vitro and in vivo and might therefore represent an effective and potentially curative therapy for extrathyroidal tumors, in particular prostate cancer.

In search for new therapeutic strategies for prostate cancer, it has been found that prostate cancer cell proliferation is modulated by androgens and a number of other factors including retinoids. Retinoids are known to play a crucial role in the control of normal epithelial cell proliferation and differentiation. They induce apoptosis and inhibit carcinogenesis, growth, invasion, angiogenesis, and metastasis of a variety of tumor types, including prostatic carcinoma cells (15, 21, 22, 23, 41, 42). Epidemiologic studies have presented evidence that retinoid deficiency plays a role in the development of prostate cancer (43, 44). Therefore, treatment with retinoids might offer a new avenue for therapy of androgen-dependent as well as androgen-independent prostate cancer. To examine the response of prostate cancer cells to retinoids, in particular regarding their proliferation, differentiation and androgen responsiveness, many investigators have used the LNCaP cell model because the growth of these cells remains androgen responsive in vitro, and many of their differentiated functions such as production of acid phosphatase and PSA are androgen regulated (23, 45, 46, 47, 48). In addition, LNCaP cells express not only high concentrations of an androgen receptor but they also have receptors for other members of the nuclear receptor family, such as retinoids (41). The action of retinoids is mediated through two families of nuclear receptors. RAR{alpha}, -ß, and -{gamma} are activated by both atRA and 9-cis-RA, and the retinoid X receptors {alpha}, ß, and {gamma} are activated by 9-cis-RA only (49, 50). All of these six forms of retinoid receptors are expressed in LNCaP cells (41).

In the current study, we examined the effect of retinoids on androgen-dependent NIS expression and therapeutic response to 131I in LNCaP cells (NP-1) that stably express the human NIS gene under the control of the PSA promoter. NIS mRNA as well as protein levels in NP-1 cells were stimulated in a concentration- and time-dependent manner following incubation with atRA in the presence of androgen, resulting in an increase of iodide accumulation mediated by RAR without affecting iodide efflux. Although no iodide organification was detected in NP-1 cells with or without atRA, the selective killing effect of 131I was increased significantly when cells were incubated with both androgen and atRA (10-7 M). In the control experiments without 131I no cytotoxic effect of atRA was detected in NP-1 cells treated with atRA in the presence or absence of androgens suggesting that the increased therapeutic effect of 131I was due to enhanced NIS protein expression and iodide accumulation. The fact that androgen-independent prostate cancer cells without PSA expression (PC-3, DU-145) did not reveal iodide uptake activity following PSA promoter-directed NIS gene transfer in the presence or absence of atRA demonstrates that atRA-induced stimulation of functional PSA promoter-directed NIS expression is selective for PSA-expressing cells and is therefore not expected to cause toxic side effects in other tissues. These data further suggest that the stimulatory effect of atRA on PSA promoter-directed NIS expression in LNCaP cells is mediated through the PSA promoter.

The growth inhibitory effects of retinoids on LNCaP cells appear to depend on a number of factors, including the passage number of the cells, concentration of RA, and presence or absence of androgens (22, 23, 28, 41, 51, 52, 53). The growth inhibition of LNCaP cells by retinoids was shown to be mediated by an androgen receptor-dependent mechanism preceded by the induction of androgen receptor gene expression (21). Also, differentiated function of prostate cells, as measured by the secretion of PSA, is enhanced by retinoids (22, 23). Other studies showed stimulation of RAR{alpha} and -{gamma} gene expression in LNCaP cells by androgens, suggesting that RAR-mediated processes might be involved in androgen regulation of prostate cells (54). In our study the stimulatory effect of atRA on androgen-induced iodide accumulation in LNCaP cells stably expressing NIS under the control of the PSA promoter was completely blocked by the androgen receptor antagonist casodex, indicating that the effect is androgen receptor mediated. AtRA-mediated stimulation of NIS gene and protein expression in LNCaP cells following PSA promoter-directed NIS gene transfer might be caused by an increase in androgen-receptor expression, thereby enhancing androgen-mediated activation of PSA promoter activity. Future studies assessing the exact interactions of androgen- and retinoid-regulated processes in prostate cancer cells are needed to fully understand the mechanisms by which atRA treatment enhances PSA promoter-directed NIS expression.

Decreased intercellular adhesion following treatment with atRA might result in enhanced NIS expression and iodide accumulation in our LNCaP cell model by increasing cell surface available for NIS protein expression and by an increased number of NIS molecules being in contact with the radioiodine-containing cell medium. Retinoic acid has been reported to affect the morphology of prostatic epithelial cells that form cell colonies containing widely spaced cells lacking intercellular adhesion when incubated in the presence of RA (42). Similar morphological changes were seen by Young et al. (51) in LNCaP cells following treatment with atRA in vitro. Further experiments are required to examine the effects of RA treatment-associated morphological changes of LNCaP cells on NIS gene and protein expression as well as iodide accumulation activity.

Potential adverse effects of androgen application in prostate cancer patients have to be considered before NIS gene therapy using the androgen-dependent PSA promoter (55). Although long-term application of androgen in these patients is certainly not recommended, exposure to androgen for only a short time period (24–48 h), which would be sufficient for PSA promoter-directed NIS gene transfer, seems to be acceptable, in particular because it serves as a prerequisite for the application of 131I, a potentially curative therapeutic approach as our previously reported data have clearly shown (12, 13, 14). In addition, in our current study, we show that treatment with atRA increases androgen-induced functional NIS expression, which not only enhances the killing effect of 131I in LNCaP cells stably expressing NIS under the control of the PSA promoter but also allows reduction of the applied androgen dose.

In conclusion, treatment with atRA increases androgen-induced functional NIS expression levels, thereby significantly enhancing the selective killing effect of 131I in LNCaP cells stably expressing NIS under the control of the PSA promoter. Thus, systemic retinoid treatment might be considered as a potent adjunct to radioiodine therapy following tissue-specific NIS gene transfer, a novel and promising cytoreductive gene therapy strategy for nonthyroidal tumors.


   Acknowledgments
 
The authors are grateful to Sissy M. Jhiang, Ph.D., Department of Physiology, Ohio State University (Columbus, OH) for supplying the full-length human NIS cDNA.


   Footnotes
 
This work was supported in part by Grant Sp 581/3-1 (to C.S.) from the German Research Council (Deutsche Forschungsgemeinschaft, Bonn, Germany); NIH Grants DK 41995 (to D.J.T.) and CA 70892 (to C.Y.F.Y.); and Mayo Foundation Prostate Cancer SPORE Grant CA91956 (to J.C.M., C.Y.F.Y., and D.J.T.).

Abbreviations: atRA, All-trans-retinoic acid; 9-cis-RA, 9-cis-retinoic acid; HBSS, Hanks’ balanced salt solution; mib, mibolerone; NIS, sodium iodide symporter; PSA, prostate-specific antigen; RAR, retinoic acid receptor; SSC, saline sodium citrate.

Received December 30, 2002.

Accepted for publication April 16, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mazzaferri EL 1996 Carcinoma of follicular epithelium: Radioiodine and other treatments and outcomes. In: Braverman LE, Utiger RD, eds. The thyroid: a fundamental and clinical text. 7th ed. Philadelphia: Lippincott-Raven; 922–945
  2. Dai G, Levy O, Carrasco N 1996 Cloning and characterization of the thyroid iodide transporter. Nature 379:458–460[CrossRef][Medline]
  3. Smanik PA, Liu Q, Furminger TL, Ryu K, Xing S, Mazzaferri EL, Jhiang SM 1996 Cloning of the human sodium iodide symporter. Biochem Biophys Res Commun 226:339–345[CrossRef][Medline]
  4. Smanik PA, Ryu K-Y, Theil KS, Mazzaferri EL, Jhiang SM 1997 Expression, exon-intron organization, and chromosome mapping of the human sodium iodide symporter. Endocrinology 138:3555–3558[Abstract/Free Full Text]
  5. Spitzweg C, Harrington KJ, Pinke LA, Vile RG, Morris JC 2001 The sodium iodide symporter and its potential in cancer therapy. J Clin Endocrinol Metab 86:3327–3335[Free Full Text]
  6. Hart IR 1996 Tissue specific promoters in targeting systemically delivered gene therapy. Semin Oncol 23:154–158[Medline]
  7. Stearns ME, McGarvey T 1992 Prostate cancer: therapeutics, diagnostics, and basic studies. Lab Invest 67:540–552[Medline]
  8. Dannull J, Belldegrun AS 1997 Development of gene therapy for prostate cancer using a novel promoter of prostate-specific antigen. Br J Urol 79:97–103
  9. Hrouda D, Dalgleish AG 1996 Gene therapy for prostate cancer. Gene Ther 3:845–852[Medline]
  10. Wei C, Willis RA, Tilton BR, Looney RJ, Lord EM, Barth RK, Frelinger JG 1997 Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: implications for tolerance and immunotherapy. Proc Natl Acad Sci USA 94:6369–6374[Abstract/Free Full Text]
  11. Cleutjens KBJM, van der Korput HAGM, Ehren-van-Eekelen CC, Sikes RA, Fasciana C, Chung LW, Trapman J 1997 A 6-kb promoter fragment mimics in transgenic mice the prostate-specific and androgen-regulated expression of the endogenous prostate-specific antigen gene in humans. Mol Endocrinol 11:1256–1265[Abstract/Free Full Text]
  12. Spitzweg C, Zhang S, Bergert ER, Castro MR, McIver B, Tindall DJ, Young CYF, Morris JC 1999 Prostate-specific antigen (PSA) promoter-driven androgen-inducible expression of sodium iodide symporter in prostate cancer cell lines. Cancer Res 59:2136–2141[Abstract/Free Full Text]
  13. Spitzweg C, O’Connor MK, Bergert ER, Tindall DJ, Young CYF, Morris JC 2000 Treatment of prostate cancer by radioiodine therapy after tissue-specific expression of the sodium iodide symporter. Cancer Res 60:6526–6530[Abstract/Free Full Text]
  14. Spitzweg C, Dietz AB, O’Connor MK, Bergert ER, Tindall DJ, Young CYF, Morris JC 2001 In vivo sodium iodide symporter gene therapy of prostate cancer. Gene Ther 8:1524–1531[CrossRef][Medline]
  15. Evans TR, Kaye SB 1999 Retinoids: present role and future potential. Br J Cancer 80:1–8[CrossRef][Medline]
  16. Kogai T, Schultz J, Johnson L, Huang M, Brent G 2000 Retinoic acid induces sodium/iodide symporter gene expression and radioiodine uptake in the MCF-7 breast cancer cell line. Proc Natl Acad Sci USA 97:8519–8524[Abstract/Free Full Text]
  17. Schmutzler C, Winzer R, Meissner-Weigl J, Kohrle J 1997 Retinoic acid increases sodium/iodide symporter mRNA levels in human thyroid cancer cell lines and suppresses expression of functional symporter in nontransformed FRTL-5 rat thyroid cells. Biochem Biophys Res Commun 240:832–838[CrossRef][Medline]
  18. Simon D, Koehrle J, Reiners C, Boerner AR, Schmutzler C, Mainz K, Goretzki PE, Roeher HD 1998 Redifferentiation therapy with retinoids: therapeutic option for advanced follicular and paillary carcinoma. World J Surg 22:569–574[CrossRef][Medline]
  19. Koshiuka K, Elstner E, Williamson E, Said JW, Tada Y, Koeffler HP 2000 Novel therapeutic approach: organic arsenical (melarsoprol) alone or with all-trans-retinoic acid markedly inhibit growth of human breast and prostate cancer cells in vitro and in vivo. Br J Cancer 82:452–458[CrossRef][Medline]
  20. Nehme A, Varadarajan P, Sellakumar G, Gerhold M, Niedner H, Zhang Q, Lin X, Christen RD 2001 Modulation of docetaxel-induced apoptosis and cell cycle arrest by all-trans-retinoic acid in prostate cancer cells. Br J Cancer 84:1571–1576[CrossRef][Medline]
  21. Zhao XY, Ly LH, Peehl DM, Feldman D 1999 Induction of androgen receptor by 1{alpha}, 25-dihydroxyvitamin D3 and 9-cis retinoic acid in LNCaP human prostate cancer cells. Endocrinology 140:1205–1212[Abstract/Free Full Text]
  22. Fong C-J, Sutkowski DM, Braun EJ, Bauer KD, Sherwood ER, Lee C, Kozlowski JM 1993 Effect of retinoic acid on the proliferation and secretory activity of androgen-responsive prostatic carcinoma cells. J Urol 149:1190–1194[Medline]
  23. Esquenet M, Swinnen JV, Heyns W, Verhoeven G 1996 Control of LNCaP proliferation and differentiation: actions and interactions of androgens, 1{alpha}, 25-dihydroxycholecalciferol, all-trans retinoic acid, 9-cis retinoic acid, and phenylacetate. Prostate 28:182–194[CrossRef][Medline]
  24. Weiss SJ, Philip NJ, Grollmann EF 1984 Iodine transport in a continuous line of cultured cells from rat thyroid. Endocrinology 114:1090–1098[Abstract/Free Full Text]
  25. Urabe M, Hershman JM, Pang X-P, Murakami S, Sugawara M 1991 Effect of lithium on function and growth of thyroid cells in vitro. Endocrinology 129:807–814[Abstract/Free Full Text]
  26. Kaminsky SM, Levy O, Salvador C, Dai G, Carrasco N 1994 Na+-I- symport activity is present in membrane vesicles from thyrotropin-deprived non-I-transporting cultured thyroid cells. Proc Natl Acad Sci USA 91:3789–3793[Abstract/Free Full Text]
  27. Castro MR, Bergert ER, Beito TG, Roche PC, Ziesmer SC, Jhiang SM, Goellner JR, Morris JC 1999 Monoclonal antibodies against the human sodium iodide symporter: utility for immunocytochemistry of thyroid cancer. J Endocrinol 163:495–504[Abstract]
  28. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  29. Spitzweg C, Joba W, Eisenmenger W, Heufelder AE 1998 Analysis of human sodium iodide symporter gene expression in extrathyroidal tissues and cloning of its complementary deoxyribonucleic acids from salivary gland, mammary gland, and gastric mucosa. J Clin Endocrinol Metab 83:1746–1751[Abstract/Free Full Text]
  30. Mandell RB, Mandell LZ, Link CJ 1999 Radioisotope concentrator gene therapy using the sodium/iodide symporter gene. Cancer Res 59:661–668[Abstract/Free Full Text]
  31. Carrasco N 1993 Iodide transport in the thyroid gland. Biochem Biophys Acta 1154:65–82[Medline]
  32. Shimura H, Haraguchi K, Myazaki A, Endo T, Onaya T 1997 Iodide uptake and experimental 131I therapy in transplanted undifferentiated thyroid cancer cells expressing the Na+/I- symporter gene. Endocrinology 138:4493–4496[Abstract/Free Full Text]
  33. Cho J-Y, Xing S, Liu X, Buckwalter TLF, Hwa L, Sferra TJ, Chiu I-M, Jhiang SM 2000 Expression and activity of human Na+/I- symporter in human glioma cells by adenovirus-mediated gene delivery. Gene Ther 7:740–749[CrossRef][Medline]
  34. Boland A, Ricard M, Opolon P, Bidart J-M, Yeh P, Filetti S, Schlumberger M, Perricaudet M 2000 Adenovirus-mediated transfer of the thyroid sodium/iodide symporter gene into tumors for a targeted radiotherapy. Cancer Res 60:3484–3492[Abstract/Free Full Text]
  35. Smit JWA, Schröder-van der Elst JP, Karperien M, Que I, van der Pluijm G, Goslings B, Romijn JA, van der Heide D 2000 Reestablishment of in vitro and in vivo iodide uptake by transfection of the human sodium iodide symporter (hNIS) in a hNIS defective human thyroid carcinoma cell line. Thyroid 10:939–943[Medline]
  36. Smit JW, Schroder-Van der Elst JP, Karperien M, Que I, Stokkel M, van der Heide D, Romijn JA 2002 Iodide kinetics and experimental (131)I therapy in a xenotransplanted human sodium-iodide symporter-transfected human follicular thyroid carcinoma cell line. J Clin Endocrinol Metab 87:1247–1253[Abstract/Free Full Text]
  37. Nakamato Y, Saga T, Misaki T, Kobayashi H, Sato N, Ishimori T, Kosugi S, Sakahara H, Konishi J 2000 Establishment and characterization of a breast cancer cell line expressing Na+/I- symporters for radioiodide concentrator gene therapy. J Nucl Med 41:1898–1904[Abstract/Free Full Text]
  38. Carlin S, Cunningham SH, Boyd M, McCluskey AG, Mairs RJ 2000 Experimental targeted radioiodide therapy following transfection of the sodium iodide symporter gene: effect on clonogenicity in both two- and three-dimensional models. Cancer Gene Ther 7:1529–1536[CrossRef][Medline]
  39. Haberkorn U, Henze M, Altmann A, Jiang S, Morr I, Mahmut M, Peschke P, Kubler W, Debus J, Eisenhut M 2001 Transfer of the human NaI symporter gene enhances iodide uptake in hepatoma cells. J Nucl Med 42:317–325[Abstract/Free Full Text]
  40. La Perle KMD, Shen D, Buckwalter TLF, Williams B, Haynam A, Hinkle G, Pozderac R, Capen CC, Jhiang SM 2001 In vivo expression and function of the sodium iodide symporter following gene transfer in the MATLyLu rat model of metastatic prostate cancer. Prostate 50:170–178
  41. Blutt SE, Allegretto EA, Pike JW, Weigel NL 1997 1, 25-dihydroxyvitamin D3 and 9-cis-retinoic acid act synergistically to inhibit the growth of LNCaP prostate cells and cause accumulation of cell in G1. Endocrinology 138:1491–1497[Abstract/Free Full Text]
  42. Peehl DM, Wong ST, Stamey TA 1993 Vitamin A regulates proliferation and differentiation of human prostatic epithelial cells. Prostate 23:69–78[Medline]
  43. Willett WC, Hunter DJ 1994 Vitamin A and cancers of the breast, large bowel, and prostate: epidemiologic evidence. Nutr Rev 52:S53–S59
  44. Reichman ME, Hayes RB, Ziegler RG, Schatzkin A, Taylor PR, Kahle LL, Fraumeni JF 1990 Serum vitamin A, and subsequent development of prostate cancer in the first National Health and Nutrition Examination Survey Epidemiologic Follow-up Study. Cancer Res 50:2311–2315[Abstract/Free Full Text]
  45. Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM, Mirand EA, Murphy GP 1983 LNCaP model of human prostatic carcinoma. Cancer Res 43:1809–1818[Abstract/Free Full Text]
  46. Berns EM, de Boer W, Mulder E 1986 Androgen-dependent growth regulation of and release of specific protein(s) by the androgen-receptor containing human prostate tumor cell line LNCaP. Prostate 9:247–259[Medline]
  47. Montgomery BT, Young CYF, Bilhartz DL, Andrews PE, Prescott JL, Thompson NF, Tindall DJ 1992 Hormonal regulation of prostate-specific antigen (PSA) glycoprotein in the human prostatic adenocarcinoma cell line, LNCaP. Prostate 21:63–73[Medline]
  48. Dahiya R, Zhang DY, Ho RJ, Haughney PC, Hayward SW, Cunha GR, Narayan P 1995 Regression of LNCaP human prostate tumor xenografts in arhymic nude mice by 13-cis-retinoic acid and androgen ablation. Biochem Mol Biol Int 35:487–498[Medline]
  49. Allegretto EA, McClurg MR, Lazarchik SB, Clemm DL, Kerner SA, Elgort MG, Boehm MF, White SK, Pike JW, Heyman RA 1993 Transactivation properties of retinoic acid and retinoid X receptors in mammalian cells and yeast. Correlation with hormone binding and effects of metabolism. J Biol Chem 268:26625–26633[Abstract/Free Full Text]
  50. Giguere V 1994 Retinoic acid receptors and cellular retinoid binding proteins: complex interplay in retinoid signaling. Endocr Rev 15:61–79[Abstract/Free Full Text]
  51. Young CYF, Murtha PE, Andrews PE, Lindzey JK, Tindall DJ 1994 Antagonism of androgen action in prostate tumor cells by retinoic acid. Prostate 25:39–45[Medline]
  52. Dahiya R, Park HD, Cusick J, Vessella RL, Fournier G, Narayan P 1994 Inhibition of tumorigenic potential and prostate-specific antigen expression in LNCaP human prostate cancer cell line by 13-cis-retinoic acid. Int J Cancer 59:126–132[Medline]
  53. Esquenet M, Swinnen JV, Heyns W, Verhoeven G 1997 LNCaP prostatic adenocarcinoma cells derived from low and high passage numbers display divergent responses not only to androgens but also retinoids. J Steroid Biochem Mol Biol 62:391–399[CrossRef][Medline]
  54. Huang HFS, Li MT, Irwin RJ Androgen and retinoic acid regulation of retinoic acid receptor (RAR) mRNA in the LNCaP cells and its relationship with cell growth. International Symposium on Biology of Prostate Growth, Washington, DC, 1996, p 80
  55. Fowler JE, Whitmore WF 1981 The response of metastatic adenocarcinoma of the prostate to exogenous testosterone. J Urol 126:372–375[Medline]



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