Purpose: Cancer specific killing can be achieved by therapeutic gene activated by cancer specific promotor. Expression of sodium iodide symporter (NIS) gene causes transportation and concentration of iodide into the cell, therefore radioiodine treatment after NIS gene transfer to cancer cell could be a form of radionuclide gene therapy. luciferase (Luc) gene transfected cancer cell can be monitored by in vivo optical imaging after D-luciferin injection. Aims of the study are to make vector with both therapeutic NIS gene driven by AFP promoter and reporter Luc gene driven by CMV promoter, to perform hepatocellular carcinoma specific radiodiodine gene therapy by the vector, and assessment of the therapy effect by optical imaging using luciferase expression. Materials and Methods: A Vector with AFP promoter driven NIS gene and CMV promoter driven Luc gene (AFP-NIS-CMV-Luc) was constructed. Liver cancer cell (HepG2, Huh-7) and non liver cancer cell (HCT-15) were transfected with the vector using liposome. Expression of the NIS gene at mRNA level was elucidated by RT-PCR. Radioiodide uptake, perchlorate blockade, and washout tests were performed and bioluminescence also measured by luminometer in these cells. In vitro clonogenic assay with 1-131 was performed. In vivo nuclear imaging was obtained with gamma camera after 1-131 intraperitoneal injection. Results: A Vector with AFP-NIS-CMV-Luc was constructed and successfully transfected into HepG2, Huh-7 and HCT-15 cells. HepG2 and Huh-7 cells with AFP-NIS-CMV-Luc gene showed higher iodide uptake than non transfected cells and the higher iodide uptake was totally blocked by addition of perchlorate. HCT-15 cell did not showed any change of iodide uptake by the gene transfection. Transfected cells had higher light output than control cells. In vitro clonogenic assay, transfected HepG2 and Huh-7 cells showed lower colony count than non transfected HepG2 and Huh-7 cells, but transfected HCT-15 cell did not showed any difference than non transfected HCT-15 cell. Number of Huh-7 cells with AFP-NIS-CMV-Luc gene transfection was positively correlated with radioidine accumulation and luciferase activity. In vivo nuclear imaging with 1-131 was successful in AFP-NIS-CMV-Luc gene transfected Huh-7 cell xenograft on nude mouse. Conclusion: A Vector with AFP promoter driven NIS and CMV promoter driven Luc gene was constructed. Transfection of the vector showed liver cancer cell specific enhancement of 1-131 cytotoxicity by AFP promoter, and the effect of the radioiodine therapy can be successfully assessed by non-invasive luminescence measurement.
Purpose: Human Na+/I- symporter (hNIS) is known to be expressed in many tissues other than thyroid gland. The breast cancer cells are one of them and the possibility of radioiodine therapy in treatment of the breast cancer may be suggested. We investigated the expression rate of hNIS and the relationship between the expression of hNIS and the finding of 99mTc-MIBI scintimammographv in the breast cancer Materials and Methods: Surgically proved 56 patients with breast cancer were the subjects of this study The expression of hNIS were evaluated by immunohistochemistry and the results were compared to the findings of 99m7c-MIBI scintimammography. Results: Overall expression rate of hNIS was 41.1% in 56 patients. According to the pathologic diagnosis, it was 42.9% in 49 patients with invasive ductal carcinoma and 28.6% in the 7 patients with ductal carcinoma in situ. The expression rate of hNIS in the 41 cases with a focal increased uptake at he breast lesion on 99m7c-MIBI sointimammogram was 31.7%. That in the 15 cases without any abnormal uptake on the scan was significantly higher(65.7%, p<0.05). Conclusion: The expression rate of hNIS in the patients with breast cancer was not so high. The rate was higher in the patients with no increased uptake at the breast lesion on 99m7c-MIBI scintimammography.
Kim, Yun-Hui;Lee, Dong-Soo;Kang, Joo-Hyun;Lee, Yong-Jin;Chung, June-Key;Lee, Myung-Chul
The Korean Journal of Nuclear Medicine
/
v.38
no.1
/
pp.99-108
/
2004
Purpose: The ability to noninvasively track the migration of neural progenitor cells would have significant clinical and research implications. We generated stably transfected F3 human neural progenitor cells with human sodium/iodide symporter (hNIS) for noninvasively tracking F3. In this study, the expression patterns of hNIS gene in F3-NIS were examined according to the cultured time and the epigenetic modulation. Materials and Methods: F3 human neural stem cells had been obtained from Dr. Seung U. Kim (Ajou University, Suwon, Korea). hNIS and hygromycin resistance gene were linked with IRES (Internal Ribosome Entry Site) under control of CMV promoter. This construct was transfected to F3 with Liposome. To investigate the restoration of hNIS gene expression in F3-NIS, cells were treated with demethylating agent (5-Azacytidine) and Histone deacetylase inhibitor (Trichostatin A: TSA). The expression of hNIS was measured by I-125 uptake assay and RT-PCR analysis. Results: The iodide uptake of the F3-NIS was higher 12.86 times than F3 cell line. According to the cell passage number, hNIS expression in F3-NIS gradually diminished. After treatment of 5-Azacytidine and TSA with serial doses (up to $20{\mu}M$, up to 62.5nM, respectively) for 24 hours, I-125 uptake and mRNA of hNIS in F3-NIS were increased. Conclusion: These results suggest that hNIS transfected F3 might undergo a change in its biological characters by cell passage. Therefore, the gene ex[ressopm of exogenous gene transferred human stem cell might be affected to the epigenetic modulation such as promoter methylation and Histone deacetylation and to the cell culture conditions.
Park, So-Yeon;Kim, Sung-Jin;Lee, Won-Woo;Lee, Heui-Ran;Kim, Hyun-Joo;Chung, June-Key;Kim, Sang-Eun
Nuclear Medicine and Molecular Imaging
/
v.42
no.5
/
pp.394-400
/
2008
Purpose: Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Materials and Methods: Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was $19.1{\pm}4.7%$, $54.0{\pm}6.4%$, $85.7{\pm}8.7%$, and $98.4{\pm}1.3%$ at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and 1-125 uptake. Results: Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro 1-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC ($29,704{\pm}6,659\; picomole/10^6\;cells$) was greater than that in adeno-hNIS-rMSC at MOI 100 ($6,168{\pm}2,134\;picomole/10^6\;cells$). Conclusion: Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.
Yoo, Ran Ji;Lee, Kyochul;Kang, Joo Hyun;Lee, Yong Jin
Journal of Radiopharmaceuticals and Molecular Probes
/
v.4
no.1
/
pp.26-31
/
2018
Macrophages play a key role in atherosclerotic plaque formation, but their participation has been discerned largely via ex vivo analyses of atherosclerotic lesions. Therefore, we aimed to identify atherosclerosis on noninvasive in vivo imaging using reporter gene system. This study demonstrated that recruitment of macrophages could be detected in atherosclerotic plaques of Apolipoprotein E knockout (ApoE-/-) mice with a sodium iodide symporter (NIS) gene imaging system using $^{99m}Tc-SPECT$. This novel approach to tracking macrophages to atherosclerotic plaques in vivo could have applications in studies of arteriosclerotic vascular disease.
Purpose: Previous studies have not showed consistent results for the level of expression of sodium/iodide symporter (NIS) in thyroid diseases, especially malignant tumor. We undertook this study to evaluate the distribution of NIS expression in malignant thyroid diseases and compare with that in benign thyroid disease. Materials and Methods: Total patients were 119 cases (Men 15, $48{\pm}13$ yrs). Total number of samples were 205 pieces. In malignant thyroid disease, there were 153 samples: 90 in papillary carcinoma, 4 in follicular carcinoma, 2 in medullary carcinoma and 57 in metastatic lymph node. In benign thyroid disease, there were 52 samples: 36 in goiter/cyst, 11 in thyroiditis and 5 in follicular adenoma. Using immunohistochemical methods, we probed 205 samples with monoclonal anti-NIS Ab. Grading of staining was stored as 0 (negative or absent), 1 (weakly positive), 2 (moderately positive) or 3 (strongly positive). Expression rate (ER) of NIS positivity in individual disease entity was expressed as percentage of total number divided by number in 2 plus 3 grade. Results: ERs of malignant thyroid diseases were 63% in papillary carcinoma, 81% in metastatic lymph node, 71% in follicular carcinoma and 100% in medullary carcinoma. ERs of benign thyroid disease were 53% in goiter/cyst, 64% in thyroiditis and 40% in follicular adenoma. ER of malignant thyroid diseases was higher than benign thyroid diseases (71% vs 54%). Grading of NIS expression in papillary carcinoma or goiter/cyst was heterogeneously distributed in considerable cases. Normal tissue also showed heterogeneous distribution of NIS expression, which was not correlated with that of primary lesion. Conclusion: In papillary thyroid carcinoma, distribution of NIS expression was heterogeneous and increased, and not different compared with that of benign thyroid disease.
Molecular nuclear cardiac imaging has included Tc-99m Annexin imaging to visualize myocardial apoptosis, but is now usually associated with gene therapy and cell-based therapy. Cardiac gene therapy was not successful so far but cardiac reporter gene imaging was made possible using HSV-TK (herpes simplex virus thymidine kinase) and F-18 FHBG (fluoro-hydroxymethylbutyl guanine) or I-124 FIAU (fluoro-deoxyiodo-arabino-furanosyluracil). Gene delivery was performed by needic injection with or without catheter guidance. Tk expression did not last longer than 2 weeks in myocardium. Cell-based therapy of ischemic heart or failing heart looks promising, but biodistribution and differentiation of transplanted cells are not known. Reporter genes can be transfected to the stem/progenitor cells and cells containing these genes can be transplanted to the recipients using catheter-based purging or injection. Repeated imaging should be available and if promoter are varied to let express reporter transgenes, cellular (trans)differentiation can be studied. NIS (sodium iodide symporter) or D2R receptor genes are promising in this aspect.
Purpose: Both human NIS and mutant $D_2R$ transgenes are proposed as reporting system in transplanted cell tracking. Using hepatoma cell lines, we constructed a dual reporter system containing human sodium-iodide symporter (hNIS) and dopamine 2 receptor ($D_2R$) and compared its characteristics. Materials and Methods: The recombinant plasmid ($pIRES-hNIS/D_2R$) was constructed with IRES (internal ribosome entry site) under control of the CMV promoter $pIRES-hNIS/D_2R$ was transfected to human hepatoma SK-Hep1 cell line with lipofectamine. HEP-ND ($SK-Hep1-hNIS/D_2R$) cells stably expressing hNIS and $D_2R$ was established by selection with G418 for two weeks. RT-PCR was performed to investigate the expression of both hNIS and $D_2R$ genes. The expressions of hNIS and $D_2R$ were measured by $^{125}I$ uptake assays and receptor binding assays. Specific binding of $D_2R$ to $[^3H]spiperone$ was verified by Scatchard plot with (+) butaclamol as a specific inhibitor. $K_d\;and\;B_{max}$ values were estimated. The correlation between hNIS and $D_2R$ expression was compared by using each clone. Results: Similar quantities of hNIS and $D_2R$ genes were expressed on HEP-ND as RT-PCR assays. HEP-ND cells showed 30 to 40 fold higher radioiodine uptakes than those of parental SK-Hep1 cells. $^{125}I$ uptake in HEP-ND cells was completely inhibited by $KClO_4$, a NIS inhibitor Specific binding to HEP-ND cells was saturable and the $K_d\;and\;B_{max}$ values for HEP-ND cells were 2.92 nM, 745.25 fmol/mg protein and 2.91nM, 1323 fmole/mg protein in two clones, respectively. The radioiodine uptake by hNIS activity and $D_2R$ binding was highly correlated. Conclusion: We developed a dual positron and gamma imaging reporter system of hNIS and $D_2R$ in a stably transfected cell line. We expect that $D_2R$ and hNIS genes can complement mutually as a nuclear reporting system or that $D_2R$ can be used as reporter gene when hNIS gene were used as a treatment gene.
Seo, Ji-Hyoung;Seong, Young-Ok;Bae, Jin-Ho;Jeong, Shin-Young;Ahn, Byeong-Cheol;Lee, Jae-Tae;Lee, Kyu-Bo
The Korean Journal of Nuclear Medicine
/
v.39
no.6
/
pp.489-490
/
2005
Purpose: The thyroglossal duct runs from the base of the tongue to the thyroid. Rarely the thyroid completely fails to migrate and results in ectopic thyroid tissue, which can be demonstrated scintigraphically. A 31-year old female patient was referred for thyroid scintigraphy due to protruding mass at the base of the tongue. She was mildly hypothyroid. Te-99m pertechnetate thyroid scan was performed to rule out ectopic thyroid gland. There showed a focal area of intense tracer uptake in sublingual area, suggesting the sublingual thyroid. In addition there noted diffusely increased tracer uptake in both breasts. The patient delivered a baby 6 months prior to the scan and was on breast-feeding. Free Tc-99m pertechnetate physiologically secrets into the salivary glands, the stomach, the gastrointestinal tract, the genitourinary tract and the mammary glands and sodium-iodide symporter plays a role in the accumulation of free Tc-99m pertechnetate. We report simultaneous visualization of lactating breasts and ectopic thyroid gland in the base of the tongue.
Serum thyroglobulin measurement and I-131 whole-body scintigraphy (WBS) are well-established methods for the detection of recurrence in the follow-up of patients with thyroid carcinoma. However, inconsistent results are observed frequently, and these two methods are not always able to detect recurrence. In some patients, serum thyroglobulin level is elevated but the WBS is negative, because the recurrent tumor is too small and below the sensitivity of the diagnostic scan, or there is a dissociation between thyroglobulin synthesis and the iodine frapping mechanism. In such cases, various nuclear imaging methods including Tl-201 Tc-99m-sestamibi, and F-18-FDG PET can be used besides anatomical imaging methods. Among them, FDG PET localizes recurrent lesions in WBS-negative thyroid carcinoma with high accuracy. Several studies have suggested that empirical high-dose I-131 therapy resulted in a high rate of visualization in post-therapy scans with evidence of subsequent improvement. An important question is when to operate on patients with recurrent tumor. We believe that surgical removal is the best means of treatment for patients with localized persistent tumor, despite the high-dose I-131 therapy. with tumor in thyroid remnant, and with isolated recurrence in the lymph node, lung or bone. In addition, we recommend palliative resection of locally unresectable mass with subsequent treatment with high-dose I-131 therapy. Before I-131 therapy, the evaluation of sodium-iodide symporter expression in thyroid carcinoma can predict iodine uptake. Retinoic acid is known to induce redifferentiation, and to enhance I-131 uptake in thyroid carcinoma. Retinoic acid therapy may represent an alternative approach before high-dose I-131 therapy.
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