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We imaged neuronal differentiation in vivo using dual reporters (sodium iodide symporter [NIS] and luciferase) coupled to a neuron-specific enolase (NSE) promoter. PC12 (NSE positive) and F11 cells were transfected with a bicistronic (NIS and luciferase; pNSE-NF) or a luciferase (pNSE-Fluc) reporter coupled to the NSE promoter. Weak NSE promoter activity was overcome by a two-step transcriptional amplification (TSTA) system (pNSE-TSTA-Fluc). In vivo, NIS and luciferase expression were examined using a 99mTc-pertechnetate gamma camera and bioluminescence imaging, respectively. pNSE-NF-transfected PC12 cells showed 3-fold higher radioiodine uptakes and >100-fold higher luciferase activity than parental cells. NIS or luciferase activity was not detected in pNSE-NF-transfected HeLa cells. When F11 cells were differentiated into neurons by db-cAMP, NIS and luciferase activities increased 4-fold compared to those without treatment, which was confirmed by Western blot and RT-PCR of NSE. In vivo in pNSE-NF-transfected F11 cells, db-cAMP treatment increased the luciferase activity but not the scintigraphic activity. In vitro, pNSE-TSTA-Fluc produced 130-fold higher luciferase activity than pNSE-Fluc and neuronal differentiation showed 4-fold higher activity from both pNSE-TSTA-Fluc and pNSE-Fluc than before differentiation. In vivo, in pNSE-TSTA-Fluc-transfected F11 cells, luciferase activity increased after neuronal differentiation. In vivo luciferase activity persisted up to 2 days after db-cAMP-induced neuronal differentiation. NSE promoter-driven dual reporter transgenes revealed the possibility of in vivo imaging of neuronal differentiation, which was further enabled by high amplification using a TSTA system. We propose that this strategy be used to follow the transplanted stem cells during differentiation in live animals. [ABSTRACT FROM AUTHOR]

Retinoic acids are natural derivatives of vitamin A, and play important roles in modulating tumor cell growth by regulating differentiation, thus suggesting the potential use of these derivatives in cancer therapy and prevention. To visualize the intranuclear responses of functional retinoic acid receptors, we have developed a dual-imaging reporter gene system based on the use of sodium/iodide symporter (NIS) and luciferase in cancer cell lines. NIS and luciferase genes were linked with an internal ribosome entry site, and placed under the control of an artificial cis-acting retinoic acid responsive element (pRARE/ NL). After retinoic acid treatment, I-125 uptake by pRARE/NL transfected cells was found to have increased by up to about five times that of nontreated cells. The bioluminescence intensity of pRARE/NL transfected cells showed dose-dependency. In vivo luciferase images showed higher intensity in retinoic acid treated SK-RARE/NL tumors, and scintigraphic images of SKRARE/NL tumors showed increased Tc-99m uptake after retinoic acid treatment. The NIS/luciferase imaging reporter system was sufficiently sensitive to allow the visualization of intranuclear retinoic acid receptor activity. This cis-enhancer imaging reporter system may be useful in vitro and in vivo for the evaluation of retinoic acid responses in such areas as cellular differentiation and chemoprevention. [ABSTRACT FROM AUTHOR]

Focuses on translational research using the sodium/iodide symporter in imaging and therapy. Role of NIS in thyroid diseases; NIS gene for radionuclide gene therapy.

To evaluate the role of p53 in radionuclide gene therapy, we investigated the cytotoxic effect of 131I and 188Re following cotransfection of the sodium iodide symporter (NIS) and wild-type p53 (wt-p53) genes into cancer cells. The NIS gene was transfected to human anaplastic thyroid carcinoma cells (ARO) expressing mutant p53 (mt-p53) using liposomes. The uptakes of 125I and 188Re were measured in the transfected (ARO-N) and wild-type cell lines (ARO). A recombinant adenovirus-5 vector containing a CMV promoter and wt-p53 cDNA, called Ad-p53, was established and transduced to ARO and ARO-N cells. After incubating cells with 131I and 188Re, the survival rate of each cell line was measured using a clonogenic assay. For radionuclide gene therapy in an animal model, Ad-p53 was injected directly into ARO and ARO-N tumours which were transplanted to nude mice. Two days later, 188Re or saline was injected intraperitoneally into the mice, and the tumours were measured using a calliper for 4 weeks. In ARO-N cells, the uptakes of 125I and 188Re were 505.16±21.30 pmol/106 cells and 13,875.20±504.85 cpm/106 cells at 30 min, respectively. There was no difference between the survival rates of ARO cells and ARO-N cells after incubation with 131I or 188Re. When Ad-p53 was transduced to ARO-N cells, the survival rate of wt-p53-expressing ARO-N cells incubated with 131I (18.5 MBq/5 ml) and 188Re (18.5 MBq/5 ml) decreased to 48.8±18.4% and 32.6±23.5%, respectively. In the nude mice experiment, ARO and ARO-N tumours gradually grew up to six to eight times larger than the initial volume. ARO and ARO-N tumours transduced with Ad-p53 continued to grow. However, the ARO-N tumours treated with Ad-p53 and 185 MBq of 188Re regressed to 20% of the initial volume. Growth of ARO-N tumour treated with 131I or 188Re was significantly inhibited by Ad-p53 transduction in vivo as well as in vitro. Transfection of the NIS gene into human anaplastic thyroid cancer induced the accumulation of beta-emitter radionuclides, and cotransfection with a wt-p53 gene enhanced the cytotoxic effect. [ABSTRACT FROM AUTHOR]

IntroductionWe conducted a molecular imaging and gene therapy method in α-fetoprotein (AFP)-producing hepatocellular carcinoma (HCC) by tumor-specific expression of the human sodiumiodide symporter (hNIS) using an AFP promoter. MethodsThe tumor-specific expression of hNIS gene by the AFP enhancerpromoter was constructed as pcDNA3-AFPhNIS. The pcDNA3-AFPhNIS was stably transfected to human HCC (Huh-7AN) and rat glioma cells (C6AN). Functional hNIS expression was confirmed by radioiodine uptake. The mRNA and protein-expression level of hNIS were measured. Biodistribution of 131I was evaluated, and scintigraphic images of 99mTc were obtained in xenografted mice. A clonogenic assay was performed by 131I. And, the in vivo therapeutic effect of 131I was evaluated in xenografted mice. ResultsIn Huh-7AN cells, iodine was highly accumulated and completely blocked by perchlorate. The protein and mRNA expression levels were correlated with iodine uptake. Radioiodine uptake in Huh-7AN tumors was higher than those of control tumors and clearly visualized. The survival rate was significantly decreased in Huh-7AN cells by 131I. Moreover, a growth of Huh-7AN tumors was inhibited by 131I in mice. ConclusionsAFP-producing hepatoma can be targeted and treated with radionuclides and hNIS, using AFP enhancerpromoter. This targeted hNIS gene therapy and molecular imaging have the potential to be used in the management of AFP-producing HCC. [ABSTRACT FROM AUTHOR]

Human telomerase reverse transcriptase (hTERT) is highly active in most cancer cells and, thus, could be used for tumor targeting. The human sodium iodide symporter (hNIS) gene is being actively researched as a potential radioactive iodine (radioiodine) gene therapy. In this study, we investigated the possibilities of using the hNIS gene driven by the hTERT promoter for molecular imaging and radioiodine gene therapy. Stable cell lines of hTERT-positive cells (Hep3B hepatoma) expressing hNIS, under the control of the 5mmTERT promoter, were generated using a retroviral system. Radioiodine uptake and efflux tests were performed, and a clonogenic assay was used to evaluate the in vitrocytotoxicity of 131I. Finally, scintigraphic, biodistribution, and radioiodine therapy studies were performed in vivo. Radioiodine uptake by 5mmTERT-NIS-transfected Hep3B cells was 22 times higher than by nontransfected Hep3B cells, and 5 times that of 5mmTERT-NIS-transfected U2-OS cells (p< 0.05). Clonogenic assays demonstrated that the survival rate of Hep3B-5mmTERT-NIS cells after 131I incubation was significantly lower than that of Hep3B cells (p< 0.001), and radioiodine accumulations in Hep3B-5mmTERT-NIS tumors were significantly higher than in wild-type tumors. In addition, technetium-99m scintigraphy clearly visualized Hep3B-5mmTERT-NIS tumors. Moreover, after being treated with 111 MBq of 131I-labeled Hep3B-5mmTERT-NIS, tumor growth was retarded, whereas Hep3B tumor growth progressed. hTERT-positive tumors were successfully targeted by the NIS gene under the control of the 5mmTERT promoter. The described system could be useful for targeted molecular imaging and as a radioiodine gene therapy for cancer. [ABSTRACT FROM AUTHOR]

Positron emission tomography (PET) imaging reporter genes, such as HSV1-tk and D2 receptor genes, make it possible to visualise gene expression non-invasively and repetitively in vivo. However, these systems require the synthesis of complicated substrates and the availability of expensive PET equipment. Expression of the sodium/iodide symporter (NIS) gene can be easily monitored with radioiodines and technetium-99m using a gamma camera. To evaluate the possibility of using NIS as an imaging reporter gene, we compared its characteristics with those of the conventional HSV1-tk gene. The CM cell line was made by transfecting the HSV1-tk gene into CT-26 (mouse colon carcinoma cell line). The CTN and CMN cell lines were then made by transfecting the NIS gene into CT-26 and CM. We measured the uptake of iodine-125 iodovinyldeoxyuridine ([125I]IVDU) and 125I to evaluate the expression of the HSV1-tk and NIS genes, respectively. Each cell line was injected into four flank sites in Balb/c mice. The biodistribution study was performed after intravenously injecting [125I]IVDU and 131I, and 131 I scintigraphy was performed for the evaluation of NIS expression. In vitro studies indicated that CTN and CMN had 40- to 79-fold and 150- to 256-fold higher uptake of 125 I than CT-26 and CM, respectively. Furthermore, CM and CMN showed 57- to 69-fold higher uptake of [125I]IVDU than CT-26 and CTN. NIS gene expression and 125I accumulation were found to be directly correlated (R 2=0.923), as were HSV1-tk gene expression and [125I]IVDU accumulation (R 2=0.956). Calculated signal per unit NIS and HSV1-tk mRNA expression was 23,2407±3,755 cpm and 34,039±5,346 cpm, re spectively. In vivo study indicated that CTN and CMN had 2.3- and 5.8-fold higher uptake of 131 I than CT-26 and CM, and 1.8- and 3.5-fold higher uptake of [125I]IVDU than CT-26 and CTN. Scintigraphy using 131I easily visualised CTN and CMN tumours. In conclusion, the NIS gene may be viewed as an imaging reporter gene with comparable performance to the HSV1-tk gene for monitoring target gene expression. [ABSTRACT FROM AUTHOR]

AbstractCell-tracking methods with molecular-imaging modality can monitor the biodistribution of cells. In this study, the direct-labeling method with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM), indirect cell-labeling methods with herpes simplex virus type 1-thymidine kinase (HSV1-tk)-mediated 124I-2?-fluoro-2?-deoxy-1-?-d-arabinofuranosyl-5-iodouracil (124I-FIAU) were comparatively investigated in vitroand in vivofor tracking of human chronic myelogenous leukemia cells. K562-TL was established by retroviral transduction of the HSV1-tkand firefly luciferasegene in the K562 cell. K562-TL cells were labeled with 64Cu-PTSM or 124I-FIAU. Cell labeling efficiency, viability, and radiolabels retention were compared in vitro. The biodistribution of radiolabeled K562-TL cells with each radiolabel and small-animal positron emission tomography imaging were performed. Additionally, in vivoand ex vivobioluminescence imaging (BLI) and tissue reverse transcriptase–polymerase chain reaction (RT-PCR) analysis were used for confirming those results. K562-TL cells were efficiently labeled with both radiolabels. The radiolabel retention (%) of 124I-FIAU (95.2%±1.1%) was fourfold higher than 64Cu-PTSM (23.6%±0.7%) at 24 hours postlabeling. Viability of radiolabeled cells was statistically nonsignificant between 124I-FIAU and 64Cu-PTSM. The radioactivity of each radiolabeled cells was predominantly accumulated in the lungs and liver at 2 hours. Both the radioactivity of 64Cu-PTSM- and 124I-FIAU-labeled cells was highly accumulated in the liver at 24 hours. However, the radioactivity of 124I-FIAU-labeled cells was markedly decreased from the body at 24 hours. The K562-TL cells were dominantly localized in the lungs and liver, which also verified by BLI and RT-PCR analysis at 2 and 24 hours postinjection. The 64Cu-PTSM-labeled cell-tracking method is more efficient than 124I-FIAU-labeled cell tracking, because of markedly decrease of radioactivity and fast efflux of 124I-FIAU in vivo. In spite of a high labeling yield and radiolabel retention of 124I-FIAU in vitro, the in vivocell-tracking method using 64Cu-PTSM could be a useful method to evaluate the distribution and targeting of various cell types, especially, stem cells and immune cells. [ABSTRACT FROM AUTHOR]

Recently, the use of a cancer deoxyribonucleic acid (DNA) vaccine encoding tumor-associated antigens has emerged as an immunotherapeutic strategy. In this study, we monitored tumor growth inhibition by pcDNA3-hMUC1 immunization in mice using optical imaging. To determine the anti-hMUC1-associated immune response generated by pcDNA3.1 or pcDNA3-hMUC1, we determined the concentration of interferon-γ (IFN-γ) protein and CD8?γ cell numbers among lymphocytes from the draining lymph nodes of mice immunized with pcDNA3.1 or pcDNA3-hMUC1. After subcutaneously injecting CT26/hMUC1-Fluc into mice immunized with pcDNA3-hMUC1, we monitored in vivo tumor growth inhibition using an optical imaging method, The concentration of IFN-γ, protein in pcDNA3-hMUC1 was higher than that of the pcDNA3,1 group [2.7 ≤ 0.08 ng/mL and 1.6 ± 0.07 ng/mL, respectively, p < .001. The number of hMUC1-associated CD8?γ cells in pcDNA3-hMUC1 immunized animals was 30-fold higher than in the pcDNA3.1 group, Bioluminescent images showed tumor growth inhibition in pcDNA3-hMUC1 immunized animals up to 25 days after immunization, A good correlation (r² = 9076: pcDNA3/hMUC1 group; r².7428: pcDNA3.1 group) was observed between bioluminescence signals and tumor weights in two mice in each group. We conclude that optical bioluminescent imaging offers a useful means of monitoring the antitumor effects of cancer DNA immunization in living animals. [ABSTRACT FROM AUTHOR]