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Transporters

 

NIS

KIM, Chung, Kang, Hwang

 

Development of a Dual Membrane Protein Reporter System Using Sodium Iodide Symporter and Mutant Dopamine D2 Receptor Transgenes

Hwang DW, Kang JH,  Chang YS, Jeong JM, Chung JK, Lee MC,  Kim S, Lee DS

Journal of Nuclear Medicine, 2007.  Vol. 48 No. 4, 588-595.

[abstract only]

 

"For noninvasive monitoring of cellular status by dual reporters, a dual membrane protein reporter system was developed and its in vivo applicability was examined. Human sodium iodide symporter (hNIS) and mutant dopamine D2 receptor (D2R) transgenes were chosen considering their complementarity.

 

Methods: pIRES-hNIS/D2R containing NIS and D2R linked with an internal ribosomal entry site (IRES) was constructed and transfected into human hepatoma SK-Hep1 and rat glioma C6 cells. The cell lines stably expressing hNIS and D2R (named SK-ND and C6-ND) were produced, which was confirmed by messenger RNA expression of reporter genes. The functional activities of hNIS and D2R were measured by 125I uptake assay and 3H-spiperone receptor-binding assays. A biodistribution study was performed on SK-ND tumor-bearing mice using 99mTc-pertechnetate and 3H-spiperone. In vivo hNIS expression was examined using 99mTc-pertechnetate {gamma}-camera imaging and, D2R expression was examined using a 3H-spiperone autoradiographic study.

 

Results: 125I uptake of SK-ND and C6-ND cell lines showed a maximum 97-fold and 43-fold increase, respectively, which were completely inhibited by KClO4. Specific 3H-spiperone binding to SK-ND and C6-ND cell homogenates was observed, which were completely inhibited by (+)-butaclamol. Among the dual reporter gene–expressing cell lines, the activities of both reporters were inversely correlated with each other. Competition assay of hNIS-expressing cells by D2R vector transfection and D2R-expressing cells by hNIS vector transfection showed a dose-dependent decrease of hNIS and D2R activities, respectively. In the biodistribution study, 99mTc-pertechnetate accumulated 10-fold and 3H-spiperone accumulated 4-fold more in SK-ND tumors than that in parental SK tumors. In vivo imaging of 99mTc-pertechnetate persisted until 5 wk after the cell graft in SK-ND tumors. Autoradiographic study of brain tissues from these mice also revealed an accumulation of 3H-spiperone in SK-ND tumors.

 

Conclusion: We developed a dual membrane-bound positron and gamma-imaging reporter system of hNIS and D2R. We observed its reporting capability in vitro and in vivo and elucidated that these 2 membrane protein reporters competed with each other in their expression. Although we expect that hNIS and D2R transgenes can complement each other as a dual reporter system, we suggest that one needs to validate the ratio of expression of the 2 membrane protein reporter transgenes for cellular status tracking."

 

 

Effect of all-trans retinoic acid on sodium/iodide symporter expression, radioiodine uptake and gene expression profiles in a human anaplastic thyroid carcinoma cell line.

Jeong H, Kim YR, Kim KN, Choe JG, Chung JK, Kim MK.

Nucl Med Biol. 2006 Oct;33(7):875-82.

 

"The plasma membrane glycoprotein sodium/iodide symporter (NIS) is crucial for thyroid hormone biosynthesis and mediates the iodide uptake of thyrocytes. It has been shown that retinoic acid (RA) alters NIS gene expression in thyroid carcinoma lines and stimulates their iodide uptake. Here, we generated an ARO human thyroidal cancer cell line that expresses the NIS gene (ARO-NIS) and found that its baseline 125I uptake was threefold higher than that of its parental ARO cells. However, a 1-microM all-trans retinoic acid (tRA) treatment significantly increased this 125I uptake up to approximately approximately 6.5-fold on Day 3. tRA also elevated NIS mRNA expression in ARO-NIS cells, with peaks of expression being observed on Day 3. To investigate the underlying genomic mechanisms involved in these tRA-induced phenotypic changes, we subjected tRA-treated and untreated ARO-NIS cells to cDNA microarray analysis. Of 1152, genes spotted onto the microarray membrane, 18 were up-regulated (z ratio>2.0) and 33 were down-regulated (z ratio<-2.0) in ARO-NIS cells after 3 days of tRA treatment. More specifically, tRA increased the expression of BCL3, CSRP3, v-fos, and CDK5 genes and decreased the expression of the FGF12 and IGFBP6 genes. Thus, tRA treatment of human anaplastic thyroid carcinoma cells stably expressing the NIS gene significantly elevates their NIS-mediated radioiodine uptake and alters the expression of many genes involved in cell growth and cellular differentiation. Therefore, tRA treatment and NIS gene transfection are potential tools for the diagnosis and treatment of thyroid cancer."

 

 

Evaluation of transcriptional activity of the oestrogen receptor with sodium iodide symporter as an imaging reporter gene.

Kang JH, Chung JK, Lee YJ, Kim KI, Jeong JM, Lee DS, Lee MC.

Nucl Med Commun. 2006 Oct;27(10):773-7.

 

"BACKGROUND: Oestrogen receptors are ligand-dependent transcription factors whose activity is modulated either by oestrogens or by an alternative signalling pathway. Oestrogen receptors interact via a specific DNA-binding domain, the oestrogen responsive element (ERE), in the promoter region of sensitive genes. This binding leads to an initiation of gene expression and hormonal effects.

 

OBJECTIVE: To determine the transcriptional activity of the oestrogen receptor, we developed a molecular imaging system using sodium iodide symporter (NIS) as a reporter gene.

 

METHODS: The NIS reporter gene was placed under the control of an artificial ERE derived from pERE-TA-SEAP and named as pERE-NIS. pERE-NIS was transferred to MCF-7, human breast cancer cells, which highly expressed oestrogen receptor-alpha with lipofectamine. Stably expressing cells were generated by selection with G418 for 14 days. After treatment of 17beta-oestradiol and tamoxifen with serial doses, the (125)I uptake was measured for the determination of NIS expression. The inhibition of NIS activity was performed with 50 micromol x l(-1) potassium perchlorate.

 

RESULTS: The MCF7/pERE-NIS treated with 17beta-oestradiol accumulated (125)I up to 70-80% higher than did non-treated cells. NIS expression was increased according to increasing doses of 17beta-oestradiol. MCF7/pERE-NIS treated with tamoxifen also accumulated (125)I up to 50% higher than did non-treated cells. Potassium perchlorate completely inhibited (125)I uptake. When MDA-MB231 cells, the oestrogen receptor-negative breast cancer cells, were transfected with pERE-NIS, (125)I uptake of MDA-MB-231/pERE-NIS did not increase.

 

CONCLUSION: This pERE-NIS reporter system is sufficiently sensitive for monitoring transcriptional activity of the oestrogen receptor. Therefore, cis-enhancer reporter systems with ERE will be applicable to the development of a novel selective oestrogen receptor modulator with low toxicity and high efficacy."

 

 

Development of a sodium/iodide symporter (NIS)-transgenic mouse for imaging of cardiomyocyte-specific reporter gene expression.

Kang JH, Lee DS, Paeng JC, Lee JS, Kim YH, Lee YJ, Hwang DW, Jeong JM, Lim SM, Chung JK, Lee MC.

J Nucl Med. 2005 Mar;46(3):479-83.

 

"Development of a small animal imaging system for differentiated cell-specific reporter gene expression will enable us to image cellular differentiation in vivo. In this study, we developed a sodium/iodide symporter (NIS)-transgenic mouse in which NIS is constitutively expressed as an imaging reporter gene only in cardiomyocytes.

 

METHODS: To express NIS gene in cardiomyocytes, alpha-myosin heavy chain (alpha-MHC)-NIS was constructed and used for the production of NIS-transgenic mice. Twelve lines of positive founder were obtained. The adequacy of the transgenic mouse model was tested by in vivo scintigraphy, microPET, and a biodistribution study.

 

RESULTS: The myocardium of transgenic mice showed rapid and intense uptake of 131I, which was much higher than that of the thyroid, and also showed long retention by gamma-camera pinhole imaging. The relative uptake ratio of the heart of transgenic mice was 4.6 +/- 1.5, which was 3.8 +/- 1.2 times higher than that of control wild-type mice. The uptake of the heart was completely blocked by oral administration of KClO4, an NIS inhibitor. The heart of transgenic mouse was also clearly and intensely visualized on microPET using 124I. Biodistribution data of these mice showed the uptake of 40-160 %ID/g (percentage injected dose per gram of tissue) of (99m)Tc-pertechnetate in the heart compared with 40-60 %ID/g in the stomach, respectively. NIS expression in the myocardium was confirmed by immunohistochemistry using a NIS-specific antibody.

 

CONCLUSION: We developed a transgenic mouse model to image cardiomyocytes with a gamma-camera and microPET using an alpha-MHC promoter and NIS. The transgenic mouse can be used as an imaging model for cardiomyocyte-specific reporter gene expression and cellular differentiation into cardiomyocytes after cardiac stem or progenitor cell transplantation."

 

 

Reversing the silencing of reporter sodium/iodide symporter transgene for stem cell tracking.

Kim YH, Lee DS, Kang JH, Lee YJ, Chung JK, Roh JK, Kim SU, Lee MC.

J Nucl Med. 2005 Feb;46(2):305-11.

 

"To track neural stem cells transfected with reporter gene, perpetual stem cell transgene expression is required. Referring to the knowledge about epigenetic modulation, we succeeded in reversing the silencing of sodium/sodide symporter (hNIS) transgenes transfected into human neural stem (HB1.F3) cells.

 

METHODS: hNIS and hygromycin resistance gene were linked with IRES (Internal Ribosome Entry Site) under control of cytomegalovirus promoter, and this construct was transfected into HB1.F3 cells to yield the F3-NIS cell lines. hNIS transgene expression was examined by (125)I uptake and reverse transcriptase polymerase chain reaction (RT-PCR). The iodide uptake of F3-NIS III cells was initially higher by up to 12.9-fold than that of nontransfected HB1.F3 cells. However, repeated passage gradually silenced hNIS expression. The recovery of hNIS transgene expression by demethylating agent (5-azacytidine) or histone deacetylase inhibitor (trichostatin A; TSA) treatment was investigated.

 

RESULTS: As hNIS transgene was gradually silenced in F3-NIS III cells, after the eighth passage its iodide uptake was 1.9-fold higher than that of nontransfected HB1.F3 cells. 5-azacytidine treatment (up to 40 micromol/L) for 24 h in F3-NIS III cells increased iodide uptake and hNIS messenger RNA (mRNA) 1.8- and 1.9-fold versus nontreated F3-NIS cells, respectively. Moreover, after TSA treatment (up to 62.5 nmol/L) for 24 h, iodide uptake and hNIS mRNA in F3-NIS III cells increased 36- and 1.9-fold versus nontreated F3-NIS III cells, respectively. The synergistic effect of demethylation and histone deacetylation inhibition was significant at high-dose 5-azacytidine and low-dose of TSA treatment. After treating F3-NIS III cells in vitro for 24 h with 62.5 nmol/L TSA, the cells were implanted into BALB/c nude mice. The TSA-treated F3-NIS III cells were clearly visible on gamma-camera imaging using (99m)Tc-pertechnetate as compared with F3-NIS III cells not treated with TSA.

 

CONCLUSION: These results suggest that 2 well-known mechanisms of epigenetic modulation synergistically are involved in silencing reporter hNIS transgene in a neural stem cell line. Transgene silencing was reversed using demethylation and histone deacetylation inhibition. We conclude that silenced reporter transgenes once successfully expressed in stem cells might be awakened by pharmacologic treatment before infusion to track stem cells in vivo."To track neural stem cells transfected with reporter gene, perpetual stem cell transgene expression is required. Referring to the knowledge about epigenetic modulation, we succeeded in reversing the silencing of sodium/sodide symporter (hNIS) transgenes transfected into human neural stem (HB1.F3) cells. METHODS: hNIS and hygromycin resistance gene were linked with IRES (Internal Ribosome Entry Site) under control of cytomegalovirus promoter, and this construct was transfected into HB1.F3 cells to yield the F3-NIS cell lines. hNIS transgene expression was examined by (125)I uptake and reverse transcriptase polymerase chain reaction (RT-PCR). The iodide uptake of F3-NIS III cells was initially higher by up to 12.9-fold than that of nontransfected HB1.F3 cells. However, repeated passage gradually silenced hNIS expression. The recovery of hNIS transgene expression by demethylating agent (5-azacytidine) or histone deacetylase inhibitor (trichostatin A; TSA) treatment was investigated.

 

 

Visualization of endogenous p53-mediated transcription in vivo using sodium iodide symporter.

Kim KI, Chung JK, Kang JH, Lee YJ, Shin JH, Oh HJ, Jeong JM, Lee DS, Lee MC.

Clin Cancer Res. 2005 Jan 1;11(1):123-8.

 

"PURPOSE: To develop a gamma camera imaging method for the determination of endogenous gene expression, we evaluated the expression of endogenous p53 gene using human sodium iodide symporter (hNIS) gene as reporter.

 

EXPERIMENTAL DESIGN: We constructed cis-p53RE-hNIS reporter vector placed under control of an artificial enhancer (p53RE). Moreover, we transfected it into human hepatoma cell line SK-Hep1 by liposome. Geneticin was used for the selection of stable transfectant (SK-Hep1p53NIS). To evaluate the function of hNIS, the inhibition study was examined with 1 mmol/L potassium perchlorate. After treatment of Adriamycin with serial dose for 24 hours, we measured the uptake of 125I and did Western blot analysis to evaluate expression of p53 protein. Tumor xenografts were produced in nude mice by s.c. injection of SK-Hep1p53NIS cells. After 7 days, scintigraphic images of nude mice before and after Adriamycin treatment were obtained using [99mTc]-pertechnetate.

 

RESULTS: In the SK-Hep1p53NIS cells, Adriamycin-treated cells accumulated up to three times higher than did nontreated cells. Potassium perchlorate inhibited completely the uptake of 125I. As Adriamycin dose increased, radioiodide uptake was significantly correlated with activated p53 as well as total p53 protein level. When Adriamycin (2 mg/kg) was treated in the same mice, a significantly higher uptake of [99mTc]-pertechnetate was observed in SK-Hep1p53NIS xenografts compared with nontreated xenografts (P < 0.05, unpaired t test).

 

CONCLUSIONS: These results suggest that p53 expression level can be monitored by NIS gene expression using cis-p53RE-hNIS system in vitro and in vivo."

 

 

In vivo imaging of retinoic acid receptor activity using a sodium/iodide symporter and luciferase dual imaging reporter gene.

So MK, Kang JH, Chung JK, Lee YJ, Shin JH, Kim KI, Jeong JM, Lee DS, Lee MC.

Mol Imaging. 2004 Jul;3(3):163-71.

[abstract only]

 

"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 SK-RARE/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."

 

 

Sodium iodide symporter: its role in nuclear medicine.

Chung JK.

J Nucl Med. 2002 Sep;43(9):1188-200. Review.

 

"Thyroid iodide uptake is basic to the clinical applications of radioiodine. Iodide uptake occurs across the membrane of the thyroid follicular cells through an active transporter process mediated by the sodium iodide symporter (NIS). The recent cloning of the NIS gene enabled the better characterization of the molecular mechanisms underlying iodide transport, thus opening the way to the clarification and expansion of its role in nuclear medicine. In papillary and follicular carcinoma, NIS immunostaining was positive in only a few tumor cells, and no NIS protein expression was detected in anaplastic carcinomas. Decreased NIS expression levels account for the reduced iodide uptake in thyroid carcinomas. Thus, by targeting NIS expression in cancer cells, we could enable these cells to concentrate iodide from plasma and in so doing offer the possibility of radioiodine therapy. Several investigators have shown that gene transfer of NIS into a variety of cell types confers increased radioiodine uptake by up to several hundredfold that of controls in nonthyroid cancers as well as in thyroid cancer. In addition, my group proposes that NIS may serve as an alternative imaging reporter gene in addition to the HSVtk and dopaminergic receptor genes. The NIS has the potential to expand the role of nuclear medicine in the future, just as it has served as the base for the development of nuclear medicine in the past."

 
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