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Codon-optimized human sodium iodide symporter (opt-hNIS) as a sensitive reporter and efficient therapeutic gene.

Kim YH, Youn H, Na J, Hong KJ, Kang KW, Lee DS, Chung JK - Theranostics (2015)

Bottom Line: The Codon Adaptation Index (CAI), representing the number of codons effective for human expression, was much improved (0.79 for hNIS, 0.97 for opt-hNIS).Both wild-type (hNIS) and optimized human NIS (opt-hNIS) were cloned into pcDNA3.1 and pMSCV vectors for transfection.Various cancer cell lines such as thyroid (TPC-1, FRO, B-CPAP), breast (MDA-MB-231), liver (Hep3B), cervical (HeLa), and glioma (U87MG) were transfected with pcDNA3.1/hNIS or pcDNA3.1/opt-hNIS. 125I uptake by opt-hNIS-expressing cells was 1.6~2.1 times higher than uptake by wild-type hNIS-expressing cells.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Nuclear Medicine, Seoul National University College of Medicine; ; 2. Biomedical Sciences, Seoul National University College of Medicine; ; 7. Division of High-Risk Pathogen Research, Korea National Institute of Health, Osong, South Korea.

ABSTRACT
To generate a more efficient in vivo reporter and therapeutic gene, we optimized the coding sequence of the human sodium/iodide symporter (NIS) gene by replacing NIS DNA codons from wild type to new codons having the highest usage in human gene translation. The Codon Adaptation Index (CAI), representing the number of codons effective for human expression, was much improved (0.79 for hNIS, 0.97 for opt-hNIS). Both wild-type (hNIS) and optimized human NIS (opt-hNIS) were cloned into pcDNA3.1 and pMSCV vectors for transfection. Various cancer cell lines such as thyroid (TPC-1, FRO, B-CPAP), breast (MDA-MB-231), liver (Hep3B), cervical (HeLa), and glioma (U87MG) were transfected with pcDNA3.1/hNIS or pcDNA3.1/opt-hNIS. 125I uptake by opt-hNIS-expressing cells was 1.6~2.1 times higher than uptake by wild-type hNIS-expressing cells. Stable cell lines were also established by retroviral transduction using pMSCV/hNIS or pMSCV/opt-hNIS, revealing higher NIS protein levels and 125I uptake in opt-hNIS-expressing cells than in hNIS-expressing cells. Moreover, scintigraphic images from cell plates and mouse xenografts showed stronger signals from opt-hNIS-expressing cells than hNIS-expressing cells, and radioactivity uptake by opt-hNIS-expressing tumors was 2.3-fold greater than that by hNIS-expressing tumors. To test the efficacy of radioiodine therapy, mouse xenograft models were established with cancer cells expressing hNIS or opt-hNIS. 131I treatment reduced tumor sizes of hNIS- and opt-hNIS-expressing tumors to 0.57- and 0.27- fold, respectively, compared to their sizes before therapy, suggesting an improved therapeutic effect of opt-hNIS. In summary, this study shows that codon optimization strongly increases hNIS protein levels and radioiodine uptake, thus supporting opt-hNIS as a more sensitive reporter and efficient therapeutic gene.

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Comparative analysis of NIS expression between cells expressing hNIS or opt-hNIS genes. (A) RT-PCR analysis of NIS transcripts in hNIS-/or opt-hNIS-expressing cells (MDA-MB-231 and FRO cells) established from monoclonal selections of retrovirus-mediated stably transduced cells. GAPDH was used as an internal control. Marked overexpression of hNIS mRNA was observed in both hNIS- and opt-hNIS-expressing cells. No expression was observed in the parental cells (CTL). (B) Western blot analysis of NIS protein in hNIS- or opt-hNIS-expressing cells. Multiple bands of glycosylated NIS were observed in both hNIS- and opt-hNIS-expressing cells, but more NIS protein was produced in opt-hNIS-expressing cells. (C) Quantitative analysis of protein expression in hNIS- or opt-hNIS-expressing cells. (D) 125I uptake assay in hNIS- or opt-hNIS-expressing cells. All experiments were performed in triplicate, and bars represent means ±SD (*, P<0.001).
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Figure 2: Comparative analysis of NIS expression between cells expressing hNIS or opt-hNIS genes. (A) RT-PCR analysis of NIS transcripts in hNIS-/or opt-hNIS-expressing cells (MDA-MB-231 and FRO cells) established from monoclonal selections of retrovirus-mediated stably transduced cells. GAPDH was used as an internal control. Marked overexpression of hNIS mRNA was observed in both hNIS- and opt-hNIS-expressing cells. No expression was observed in the parental cells (CTL). (B) Western blot analysis of NIS protein in hNIS- or opt-hNIS-expressing cells. Multiple bands of glycosylated NIS were observed in both hNIS- and opt-hNIS-expressing cells, but more NIS protein was produced in opt-hNIS-expressing cells. (C) Quantitative analysis of protein expression in hNIS- or opt-hNIS-expressing cells. (D) 125I uptake assay in hNIS- or opt-hNIS-expressing cells. All experiments were performed in triplicate, and bars represent means ±SD (*, P<0.001).

Mentions: To remove the effect of variable factors from transient transfection such as transfection efficiency and copy number variation, stable FRO and MDA-MB-231 cell lines were established by retroviral transduction using pMSCV/hNIS or pMSCV/opt-hNIS. Since the natural NIS protein is located in the basolateral membrane of normal thyroid and lactating mammary glands, FRO and MDA-MB-231 cells were selected as representatives for thyroid and breast cancer. By real-time quantitative PCR, monoclonal FRO or MDA-MB-231 cells expressing similar number of hNIS mRNA transcripts were selected to evaluate functional hNIS production. NIS mRNAs were clearly detected at a similar level in both hNIS- and opt-hNIS-expressing cells (Figure 2A). The NIS protein appeared as multiple bands consisting of its major unglycosylated (72 kDa) and glycosylated (97 kDa or higher) forms in both hNIS- and opt-hNIS-transduced cells (Figure 2B). However, more NIS protein was produced in opt-hNIS-expressing cells than in hNIS-expressing cells. Quantitative analysis of western blot data verified that opt-hNIS protein expression increased 4- to 5.4-fold over that of wild-type hNIS (Figure 2C). 125I uptake by opt-hNIS-expressing cells was also 2.4 to 3.1 times higher than uptake by hNIS-expressing cells (Figure 2D).


Codon-optimized human sodium iodide symporter (opt-hNIS) as a sensitive reporter and efficient therapeutic gene.

Kim YH, Youn H, Na J, Hong KJ, Kang KW, Lee DS, Chung JK - Theranostics (2015)

Comparative analysis of NIS expression between cells expressing hNIS or opt-hNIS genes. (A) RT-PCR analysis of NIS transcripts in hNIS-/or opt-hNIS-expressing cells (MDA-MB-231 and FRO cells) established from monoclonal selections of retrovirus-mediated stably transduced cells. GAPDH was used as an internal control. Marked overexpression of hNIS mRNA was observed in both hNIS- and opt-hNIS-expressing cells. No expression was observed in the parental cells (CTL). (B) Western blot analysis of NIS protein in hNIS- or opt-hNIS-expressing cells. Multiple bands of glycosylated NIS were observed in both hNIS- and opt-hNIS-expressing cells, but more NIS protein was produced in opt-hNIS-expressing cells. (C) Quantitative analysis of protein expression in hNIS- or opt-hNIS-expressing cells. (D) 125I uptake assay in hNIS- or opt-hNIS-expressing cells. All experiments were performed in triplicate, and bars represent means ±SD (*, P<0.001).
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Figure 2: Comparative analysis of NIS expression between cells expressing hNIS or opt-hNIS genes. (A) RT-PCR analysis of NIS transcripts in hNIS-/or opt-hNIS-expressing cells (MDA-MB-231 and FRO cells) established from monoclonal selections of retrovirus-mediated stably transduced cells. GAPDH was used as an internal control. Marked overexpression of hNIS mRNA was observed in both hNIS- and opt-hNIS-expressing cells. No expression was observed in the parental cells (CTL). (B) Western blot analysis of NIS protein in hNIS- or opt-hNIS-expressing cells. Multiple bands of glycosylated NIS were observed in both hNIS- and opt-hNIS-expressing cells, but more NIS protein was produced in opt-hNIS-expressing cells. (C) Quantitative analysis of protein expression in hNIS- or opt-hNIS-expressing cells. (D) 125I uptake assay in hNIS- or opt-hNIS-expressing cells. All experiments were performed in triplicate, and bars represent means ±SD (*, P<0.001).
Mentions: To remove the effect of variable factors from transient transfection such as transfection efficiency and copy number variation, stable FRO and MDA-MB-231 cell lines were established by retroviral transduction using pMSCV/hNIS or pMSCV/opt-hNIS. Since the natural NIS protein is located in the basolateral membrane of normal thyroid and lactating mammary glands, FRO and MDA-MB-231 cells were selected as representatives for thyroid and breast cancer. By real-time quantitative PCR, monoclonal FRO or MDA-MB-231 cells expressing similar number of hNIS mRNA transcripts were selected to evaluate functional hNIS production. NIS mRNAs were clearly detected at a similar level in both hNIS- and opt-hNIS-expressing cells (Figure 2A). The NIS protein appeared as multiple bands consisting of its major unglycosylated (72 kDa) and glycosylated (97 kDa or higher) forms in both hNIS- and opt-hNIS-transduced cells (Figure 2B). However, more NIS protein was produced in opt-hNIS-expressing cells than in hNIS-expressing cells. Quantitative analysis of western blot data verified that opt-hNIS protein expression increased 4- to 5.4-fold over that of wild-type hNIS (Figure 2C). 125I uptake by opt-hNIS-expressing cells was also 2.4 to 3.1 times higher than uptake by hNIS-expressing cells (Figure 2D).

Bottom Line: The Codon Adaptation Index (CAI), representing the number of codons effective for human expression, was much improved (0.79 for hNIS, 0.97 for opt-hNIS).Both wild-type (hNIS) and optimized human NIS (opt-hNIS) were cloned into pcDNA3.1 and pMSCV vectors for transfection.Various cancer cell lines such as thyroid (TPC-1, FRO, B-CPAP), breast (MDA-MB-231), liver (Hep3B), cervical (HeLa), and glioma (U87MG) were transfected with pcDNA3.1/hNIS or pcDNA3.1/opt-hNIS. 125I uptake by opt-hNIS-expressing cells was 1.6~2.1 times higher than uptake by wild-type hNIS-expressing cells.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Nuclear Medicine, Seoul National University College of Medicine; ; 2. Biomedical Sciences, Seoul National University College of Medicine; ; 7. Division of High-Risk Pathogen Research, Korea National Institute of Health, Osong, South Korea.

ABSTRACT
To generate a more efficient in vivo reporter and therapeutic gene, we optimized the coding sequence of the human sodium/iodide symporter (NIS) gene by replacing NIS DNA codons from wild type to new codons having the highest usage in human gene translation. The Codon Adaptation Index (CAI), representing the number of codons effective for human expression, was much improved (0.79 for hNIS, 0.97 for opt-hNIS). Both wild-type (hNIS) and optimized human NIS (opt-hNIS) were cloned into pcDNA3.1 and pMSCV vectors for transfection. Various cancer cell lines such as thyroid (TPC-1, FRO, B-CPAP), breast (MDA-MB-231), liver (Hep3B), cervical (HeLa), and glioma (U87MG) were transfected with pcDNA3.1/hNIS or pcDNA3.1/opt-hNIS. 125I uptake by opt-hNIS-expressing cells was 1.6~2.1 times higher than uptake by wild-type hNIS-expressing cells. Stable cell lines were also established by retroviral transduction using pMSCV/hNIS or pMSCV/opt-hNIS, revealing higher NIS protein levels and 125I uptake in opt-hNIS-expressing cells than in hNIS-expressing cells. Moreover, scintigraphic images from cell plates and mouse xenografts showed stronger signals from opt-hNIS-expressing cells than hNIS-expressing cells, and radioactivity uptake by opt-hNIS-expressing tumors was 2.3-fold greater than that by hNIS-expressing tumors. To test the efficacy of radioiodine therapy, mouse xenograft models were established with cancer cells expressing hNIS or opt-hNIS. 131I treatment reduced tumor sizes of hNIS- and opt-hNIS-expressing tumors to 0.57- and 0.27- fold, respectively, compared to their sizes before therapy, suggesting an improved therapeutic effect of opt-hNIS. In summary, this study shows that codon optimization strongly increases hNIS protein levels and radioiodine uptake, thus supporting opt-hNIS as a more sensitive reporter and efficient therapeutic gene.

Show MeSH
Related in: MedlinePlus