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Basic Science Investigation |
1 Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, Korea; 2 Institute of Radiation Medicine, Seoul National University College of Medicine, Seoul, Korea; 3 Tumor Immunity Medical Research Center, Seoul National University College of Medicine, Seoul, Korea; and 4 Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea
Correspondence: For correspondence or reprints contact: June-Key Chung, MD, PhD, Department of Nuclear Medicine, Seoul National University Hospital, 28, Yongon-dong, Jongno-gu, Seoul 110-744, Korea. E-mail: jkchung{at}plaza.snu.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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B protein were executed. Electrophoretic mobility-shift assay (EMSA) was performed to evaluate nuclear factor-B (NF-B) binding activity induced by doxorubicin. Scintigraphic and bioluminescent images were acquired and quantitated before and after doxorubicin in a tumor-bearing mouse model. Results: Radioiodide uptake in ARO cells transfected with the NIS gene under the CMV promoter was remarkably enhanced by doxorubicin, and this corresponded to a significant increase in NIS messenger RNA. In addition, luciferase gene upregulation by doxorubicin was also observed in luciferase gene transfected ARO cells. These results were verified by in vivo imaging in a tumor-bearing mouse model. Moreover, transgene expressional enhancement by doxorubicin was observed after transfecting ARO cells with adenoviral vector or plasmid vector, when transgenes were under the control of a CMV promoter. In addition, NF-B, activated by doxorubicin, induced transgene transcription under the control of the CMV promoter, which possesses an NF-B binding site. Conclusion: These findings indicate that doxorubicin enhances transgene expression under the control of the CMV promoter and that doxorubicin might be used as an adjuvant to radioiodine therapy by NIS gene transfer in anaplastic thyroid carcinoma.
Key Words: anaplastic thyroid cancer • doxorubicin • transgene • imaging reporter gene • gene therapy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Sodium/iodide symporter (NIS) is a specific transporter that cotransports sodium and an iodide ion. The targeted expression of NIS in cancer cells allows these cells to concentrate iodide from plasma, and several radiotargeted gene therapy studies using NIS gene transfer into various cancer cells and tumor xenografts have achieved encouraging results (5–7). In addition, the recently cloned and characterized human NIS (hNIS) gene has attracted attention because of its strong iodide transport activity, nonimmunoreactivity, and proven efficiency in the radioiodine therapy of thyroid cancer (8–10). However, despite the successes of these investigations, improved gene expression strategies are needed to enhance the efficacies of gene therapies.
Previously, we transferred the NIS gene under cytomegalovirus (CMV) promoter into human anaplastic thyroid cancer cells (ARO cells) for radioiodine therapy and therapeutic intervention imaging (11). However, several problems remain, such as rapid washout of radionuclide from cells and its limited retention within cells (9). Thus, new strategies are needed to enhance radionuclide accumulation or retain radioiodine in cells. Here, we report that radioiodide uptake was remarkably enhanced when doxorubicin was administered to hNIS gene–transfected ARO cells. Genotoxic stresses, such as those induced by irradiation and chemotherapy, are known to enhance transgene expression, including NF-B activation, in cancer cells (12,13), and it has been reported that CMV promoter, a strong viral promoter, has consensus binding sites for NF-B in its enhancer region (14).
In this study, transgene expression under the control of a CMV promoter was enhanced in ARO cells in vitro and in a mouse model by doxorubicin. This enhancement was mediated by the activation of NF-B in the CMV promoter. Overall, our results provide a useful strategy for radioiodine therapy by NIS gene transfer in anaplastic thyroid carcinoma and a means for improving therapeutic gene expression in other gene therapy applications.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Adenoviruses expressing hNIS (Ad-hNIS) or luciferase (Ad-Luc) under the control of the CMV promoter were made by recombining hNIS containing shuttle vector or luciferase containing shuttle vector with adenoviral backbone, respectively, using the AdEasy Adenoviral Vector System, following the manufacturer's instructions (Qbiogene). Viruses were amplified in 293 cells, purified by CsCl gradient ultracentrifugation, and then titered using the median tissue culture infective dose (TCID50) according to standard protocols. Viral stocks were maintained at –70°C for long-term storage.
Cell Culture and Vector Transfer
Human anaplastic thyroid cancer cells, ARO cells, were grown in RPMI-1640 medium containing 100,000 IU/L penicillin, 100 µg/mL streptomycin, 250 µg/L amphotericin B, and 10% (v/v) fetal bovine serum (Invitrogen) in a humidified 5% CO2/95% air atmosphere at 37°C. Cell viability was evaluated by trypan blue exclusion.
ARO cells were transfected with pCMV-NIS or pCMV-Luc vector using Lipofectamine PLUS reagent, according to the manufacturer's instructions (Invitrogen). Selection was performed using 300–800 µg/mL geneticin (Invitrogen) in RPMI-1640 medium containing 10% fetal bovine serum for 2 wk, beginning the day after transfection. Surviving clones (ARO-hNIS and ARO-Luc) were isolated and screened for iodide uptake activity and bioluminescence.
In addition, ARO cells were transduced at a multiplicity of infection of 0.1 plaque-forming unit (pfu) with Ad-hNIS and Ad-Luc, respectively. Vector was then removed by washing, and cells were further cultured for 24 h before being analyzed for transgene expression.
Treatments with Doxorubicin or NF-B Pathway Inhibitor
Transduced and control ARO cells were inoculated in quadruplicate into 24-well plates at 3 x 105 cells per well. After incubation for 12 h, each well was washed with phosphate-buffered saline (PBS), and medium was replaced with fresh medium containing doxorubicin at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 µg/mL. The cells were then incubated in 5% CO2 for 24 h at 37°C. For NF-B pathway inhibition assays, cells were pretreated with the inhibitory B kinase (IKK) inhibitor 10 µM Bay 11-7082 (Alexis), for 1 h, and then incubated in the presence of doxorubicin.
125I Uptake Assays
ARO cells stably transfected with reporter vector (ARO-hNIS) were plated at 3 x 105 cells per well in a 24-well plate and then cultured for 12 h with RPMI-1640 medium containing 10% fetal bovine serum. The cells were then treated with doxorubicin, and 24 h later clones were assessed for NIS activity by measuring radioiodide uptake at 37°C using a previously described method (15). In brief, iodide uptake levels were determined by incubating cells with 500 µL Hanks' balanced salt solution (HBSS) containing 0.5% bovine serum albumin (BSA) and 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid/NaOH (pH 7.4), 3.7 kBq of carrier-free Na125I, and 10 µM NaI, to yield a specific activity of 740 MBq/mmol at 37°C over 30 min. After incubation, cells were washed twice, as quickly as possible (<15 s), with 2 mL of iodine-free ice-cold HBSS buffer, detached with 500 µL trypsin, and then measured for radioactivity using a 
-counter (Cobra II; Canberra Packard).
Luciferase Assays
Transfected and control ARO cells were inoculated into 24-well plates at 3 x 105 cells per well. After incubation for 12 h, wells were washed with PBS, medium was replaced with fresh medium containing various concentrations of doxorubicin, and cells were incubated in 5% CO2 for 24 h at 37°C. Bioluminescence assays were performed using Luciferase Assay Kits (Applied Biosystems). Lysis solution was added to each well, and cell lysates were transferred to microplates. Bioluminescence was measured using a TR717 microplate luminometer (Applied Biosystems).
For in vitro bioluminescent imaging, after adding a mixture of luciferin and buffer solution, microplates were placed in a dark box of the LAS-3000 luminescent image analyzer (Fuji Film), and images were acquired over 2 min. Acquired bioluminescent images were quantified using the Multi Gauge, version 3.02, image analysis program (Fuji Film), which determined the optical densities (ODs) of regions of interest (ROIs) on the images.
Reverse Transcriptase Polymerase Chain Rreaction (RT-PCR) Analysis for hNIS
Total RNA was prepared from ARO-NIS cells using TRIZOL reagent (Invitrogen) according to the manufacturer's instructions, with slight modification. Briefly, approximately 500 ng of total RNA were reverse-transcribed in a final volume of 20 µL containing 1 µL oligo(dT), 4 µL 5x first-strand buffer, 2 µL 0.1 M dithiothreitol (DTT), 1 µL 10 mM deoxyribonucleoside triphosphate (dNTP) mix, and Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Invitrogen). PCR was performed in a total volume of 20 µL containing 2 µL of complementary DNA, 2 µL of 10x reaction buffer, forward primer (5'-TCTCTCAGTCAACGCCTCT-3') and reverse primer (5'-ATCCAGGATGGCCACTTCTT-3'), 1 µL of 10 mM dNTP, and 2.5 units of Taq-DNA polymerase (GeneCraft) using a GeneAmp PCR System (Applied Biosystems). Samples were subjected to 5 min of denaturation at 94°C, 25 amplification cycles (30 s at 94°C, 30 s at 47°C, and 1 min at 72°C), and an additional 5 min at 72°C.
ß-Actin was amplified as a control using the same reaction conditions but with ß-actin primers, forward (5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3') and reverse (5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'). Samples were subjected to 5 min of denaturation at 94°C, 23 amplification cycles (30 s at 94°C, 30 s at 50°C, and 1 min at 72°C), and an additional 5 min at 72°C. Amplified products were analyzed by ethidium bromide–stained agarose gel electrophoresis. Results were interpreted using Tina 2.1 software (Raytest). Expression values of the NIS gene were calculated by dividing NIS band intensities by those of ß-actin. The correlation between gene expression (by RT-PCR) and protein activity (by radioiodide uptake) was analyzed using Sigma Plot 2001 (SPSS Inc.).
Western Blot Analysis
For the immunodetection of IB
, ARO-hNIS cells were incubated with 0, 1, 2, 3, 4, or 5 µg/mL of doxorubicin for 24 h, and cytoplasmic extracts were then prepared. Cells were harvested with a scraper after being washed with ice-cold PBS and lysed in a buffer containing 10 mM Tris·HCl (pH 7.5), 1 mM DTT, 20% (v/v) glycerol, 1 mM ethylenediaminetetraacetic acid (EDTA), and a protease inhibitor mixture. Samples were then centrifuged at 4°C for 5 min, and supernatants were mixed with NaDodSO4 sample buffer (1:2), and boiled for 5 min. Samples (10 µL) of these mixtures were electrophoresed in bis-Tris·HCl buffered 4%–12% gradient polyacrylamide gels (Invitrogen). After transferring proteins to nitrocellulose membranes (Schleicher & Schuell Inc.) by electroblotting, membranes were incubated in 3% skim milk in TBS-T (20 mM Tris, 137 mM NaCl, and 0.1% Tween 20), and then treated with antibodies against IB (sc-371; dilution, 1:1,000; Santa Cruz Biotechnology), and -tubulin (clone B-5-1-2; dilution, 1:2,000; Sigma). These proteins were located using enhanced chemiluminescence (ECL) reagents (Roche). Protein concentrations in samples were assayed using BCA Protein Assay kits (Pierce).
Electrophoretic Mobility-Shift Assays (EMSA)
Nuclear extracts were prepared from doxorubicin-nontreated and -treated (at 3 µg/mL) ARO-hNIS cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents kits, according to the manufacturer's instructions (Pierce). Gel-shift assays were performed using EMSA kits (Panomics). For the binding reaction, 5 µg of nuclear extracts were incubated at room temperature for 30 min with reaction buffer containing; 20 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) at pH 7.9, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 5% glycerol, 200 µg/mL BSA, 2 µg of poly(dI-dC), and the biotin-labeled probes (10 ng), containing the NF-B binding consensus sequence (5'-AGTTGAGGGGACTTTCCCAGGC-3'). The negative control contained free probe (without nuclear extract).
To further prove binding specificity, a competition experiment was performed with an unlabeled oligonucleotide containing the NF-B binding site of the CMV promoter. As a competition control, excess unlabeled cold probes were added to samples containing nuclear extract and biotin-labeled probe. Samples were separated in 6% polyacrylamide gels in 0.5% Tris-borate-EDTA (TBE) and transferred onto nylon membranes, which were fixed by ultraviolet-induced cross-linking. The biotin-labeled probe was detected using streptavidin/horseradish peroxidase (Panomics).
Animal Experiments
All animal experiments were performed with the approval of the Seoul National University Animal Research Committee. Tumor xenografts were produced in male BALB/c nude mice, 6-wk-old, by subcutaneously injecting 5 x 106 ARO cells suspended in 100 µL of serum-free RPMI-1640 medium into both left and right hind flanks. After allowing 2 wk for tumor growth, when the diameter of tumors reached a minimum size of 7 mm, adenoviruses were injected. PBS and Ad-hNIS (1 x 107 pfu) were injected into tumor masses in the left and right thighs, respectively. For bioluminescent imaging, Ad-Luc (1 x 107 pfu) was injected instead of Ad-hNIS. Because the adenoviral vector is not designed for cancer cell-specific infection, the vector was injected intratumorally.
On day 2 after adenovirus injection, scintigraphic and bioluminescent images were acquired. Anesthesia was performed by intraperitoneally injecting ketamine (53 mg/kg) and xylazine (12 mg/kg) solution 10 min before imaging.
To obtain scintigraphic images, 30 min after injecting 18.5 MBq of 99mTc-pertechnetate intraperitoneally per animal, mice were placed in a spread prone position and scanned with an ON 410 -camera (Ohio Nuclear) equipped with a pinhole collimator. Chen et al. (16) demonstrated that treatment effects were not dependent on the administration route (intraperitoneal and intravenous). We chose the intraperitoneal injection method, which is more easily performed and reproducible than the intravenous injection method. To estimate tumor dosimetry and to confirm scintigraphic measurements, individual tumors and muscle were sampled, weighed, and assayed for 99mTc-pertechnetate radioactivity using a -counter (Cobra II; Canberra Packard). To normalize data to body background activity, tumor-to-muscle radioactivity ratios were calculated.
For bioluminescent imaging, an aqueous solution of luciferin (5 mg/mouse; Molecular Probes) was injected intraperitoneally over 10 min, and then the animal was placed in an LAS-3000 dark box (Fuji Film). After obtaining gray-scale images under white light, bioluminescent images were acquired by collecting and integrating light for 10 min. Image processing and analysis were performed as described earlier. Acquired bioluminescent images were quantified using the Multi Gauge, version 3.02 image analysis program (Fuji Film), which determined the ODs of ROIs on images.
One day after a 0.2-mL intraperitoneal injection of 3.2 mg/kg doxorubicin, scintigraphic and bioluminescent images were also obtained as described.
Statistical Analysis
Results are expressed as mean ± SD. The unpaired t test was used to determine the statistical significance for all comparisons. P values < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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When ARO-Luc cells were treated with doxorubicin, bioluminescence increased markedly in the same manner as that observed in ARO-hNIS cells (Fig. 2). At 3 µg/mL, bioluminescence was maximal at 13.3-fold; further increases in doxorubicin reduced bioluminescence (5.2-fold at 4 µg/mL and 1.4-fold at 5 µg/mL).
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Induction of Transgene Expression by Doxorubicin in ARO Cells Transduced with Adenoviral Vectors
We also analyzed whether transgene expression by adenoviral vector was induced by doxorubicin. A dramatic increase in iodide uptake and bioluminescence was observed after administering doxorubicin to transduced ARO cells, where hNIS and Luc genes were under the control of the CMV promoter in adenoviral vector (Figs. 3 and 4). When Ad-hNIS–transduced ARO cells were treated with doxorubicin for 24 h, iodide uptake levels significantly increased (3.7-fold at 3 µg/mL) (Fig. 3). Ad-Luc–transduced ARO cells also showed peak (24.5-fold) luciferase activity at a doxorubicin concentration of 3 µg/mL (Fig. 4).
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B Activity on CMV PromoterB participates in the effect of doxorubicin on enhancement of transgene expression, IB protein expression was detected by Western blotting using an anti-IB antibody. When the cytoplasmic extracts of ARO-hNIS cells treated with 0, 1, 2, 3, 4, or 5 µg/mL of doxorubicin were examined, significant dose-dependent reductions in immunoreactive IB protein levels were observed (Fig. 5A).
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B binding to biotin-labeled oligonucleotide after 24 h of doxorubicin treatment (Fig. 5B). Competition with unlabeled oligonucleotide containing the NF-B binding site of the CMV promoter resulted in a significant reduction in NF-B binding. However, biotin-labeled oligonucleotide without nuclear extracts showed no specific binding activity for NF-B.
We also pretreated transduced ARO cells with the IKK inhibitor (Bay 11-7082) before adding doxorubicin and then examined whether the enhancement of transgene expression by doxorubicin was selectively targeted for the activation of the NF-B pathway. Treatment with doxorubicin resulted in an increase in iodide uptake or bioluminescence in Ad-hNIS– or Ad-Luc–infected ARO cells, respectively. Moreover, as expected, treatment with the IKK inhibitor markedly inhibited the induction of transgene expression by doxorubicin (Fig. 6; **P < 0.005 vs. doxorubicin-treated cells infected with Ad-hNIS or Ad-Luc).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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B response element of CMV promoter, the combination of doxorubicin and radioiodine could be a more useful strategy for cancer therapy. In the present study, we observed that doxorubicin enhanced transgene expression under the control of the CMV promoter in vitro and in vivo. When ARO-hNIS cells were treated with doxorubicin, iodide uptake significantly increased up to 21.9-fold and, when mice bearing different types of tumors were treated with doxorubicin, significantly higher iodide uptake or bioluminescence was observed in Ad-hNIS– or Ad-Luc–infected ARO xenografts, respectively, than in wild-type ARO xenografts or in nontreated Ad-hNIS or Ad-Luc–infected ARO xenografts. RT-PCR analysis also suggested that this increased transgene expression is the result of increased transgene mRNA production.
In our study, transfected and transduced ARO cells showed maximal transgene expressions and activities at a doxorubicin concentration of 3 µg/mL; further increases in doxorubicin reduced, rather than enhanced, transgene expression (Fig. 1B). Doxorubicin exerts its cytotoxicity by inducing DNA strand breaks by interacting with topoisomerase II (26,27), and this effect of doxorubicin can induce cell cycle arrest or apoptosis. As shown in Figure 1A inset, ARO cells maintained viability even at a doxorubicin concentration of 20 µg/mL. We observed that the population of doxorubicin (5 µg/mL)–treated ARO cells remained constant for 48 h (data not shown). These results suggest that the transcription of transgene was abruptly inactivated by the stalling of RNA polymerase II complexes at doxorubicin concentrations exceeding 3 µg/mL in ARO-hNIS and ARO-Luc cells under cell cycle arrest by doxorubicin treatment (28). Actually, in cell cycle analysis according to doxorubicin concentration, transient G2/M arrest occurred up to 2 µg/mL, and G1 arrest progressed with concentrations of 3 µg/mL or higher. Our data are concordant with the demonstration of O'Loughlin et al. (29) in which transient G2/M arrest and subsequent progression into G1 occurred dependent on doxorubicin exposure time in drug-resistant carcinoma cells.
When 125I uptake was measured in doxorubicin-treated ARO cells versus nontreated cells, no significant difference was observed. This result suggests that doxorubicin does not reactivate the silenced endogenous NIS gene but, rather, it induces transgene activation under the control of the CMV promoter. This effect of doxorubicin was also verified in ARO cells transduced with adenoviral vectors, including the CMV promoter.
Anthracycline drugs, such as doxorubicin, are known to be able to induce NF-B activation (13), which results from the phosphorylation-induced degradation of its inhibitory protein, IB, by proteosome (30,31). It is generally accepted that the activation of the NF-B pathway in cancer cells represents an antiapoptotic mechanism (32,33). Eventually, the activation of this pathway can make cancer cells resistant to chemotherapy and radiotherapy. In the present study, we found that doxorubicin stimulated the activation of NF-B and its binding to the response element localized in the CMV promoter region and induced transgene expression, as was shown by the degradation of IB by Western blotting and NF-B binding by EMSA. In this study, the NF-B pathway activated by doxorubicin induced NIS gene expression, resulting in the accumulation of therapeutic radioiodine. This finding indicates that doxorubicin pretreatment before radioiodine gene therapy may be effective for treating cancers that are resistant to conventional chemotherapy or radiotherapy.
However, we do not believe that this response of transgene expression is limited only to doxorubicin because it has been reported that most anticancer drugs, as DNA-damaging agents, activate the NF-B pathway. In our study, it was shown that the CMV promoter containing the NF-B response element in its enhancer region was triggered by NF-B pathway activation as a result of doxorubicin treatment. Therefore, when other promoters have the NF-B response element, these promoters can be activated by doxorubicin.
Our study shows that doxorubicin significantly enhances transgene expression under the control of a CMV promoter both in vitro and in vivo. In addition, we demonstrate that this enhancement is mediated by the activation of NF-B. Moreover, because the CMV promoter has cis-regulatory elements recognized by various transcription factors—such as AP-1 (34), CREB (35), retinoic acid nuclear receptors (RARs) (36), and NF-B (37)—further studies of other factors and combination treatments are required to help elucidate the reasons for improved transgene activity. Further experiments evaluating a prolonged accumulation of radionuclide and the tumorcidal effect of this improved method are also needed. Nevertheless, our results provide a useful strategy for gene therapy in the treatment of cancers in general.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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B in the CMV promoter. These findings indicate that doxorubicin might be used as an adjuvant to radioiodine therapy by NIS gene transfer in anaplastic thyroid carcinoma. Further study in various cancers could provide a useful strategy for cancer gene therapy. |
ACKNOWLEDGMENTS |
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FOOTNOTES |
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References |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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B by antineoplastic agents: role of protein kinase C. J Biol Chem. 1997;272:14914–14920. B activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T cell stimulation. EMBO J. 1989;8:4251–4258.[Medline]Related articles in JNM:
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