Efficient multicistronic co-expression of hNIS and hTPO in prostate cancer cells for nonthyroidal tumor radioiodine therapy

Abstract

Radioiodine therapy has proven to be a safe and effective approach in the treatment of differentiated thyroid cancer. Similar treatment strategies have been exploited in nonthyroidal malignancies by transfecting hNIS gene into tumor cells or xenografts. However, rapid radioiodine efflux is often observed after radioiodine uptake, limiting the overall antitumor effects. In this study, we aimed at constructing multicistronic co-expression of hNIS and hTPO genes in tumor cells to enhance the radioiodine uptake and prolong the radioiodine retention. Driven by the cytomegalovirus promoter, hNIS and hTPO were simultaneously inserted into the expression cassette of adenoviral vector. An Ad5 viral vector (Ad-CMV-hTPO-T2A-hNIS) was assembled as a gene therapy vehicle by Gateway technology and 2A method. The co-expression of hNIS and hTPO genes was confirmed by a double-label immunofluorescence assay. The radioiodine ((125)I) uptake and efflux effects induced by co-expression of hNIS and hTPO genes were determined in transfected and non-transfected PC-3 cells. Significantly higher uptake (6.58 ± 0.56 fold, at 1 h post-incubation) and prolonged retention (5.47 ± 0.36 fold, at 1 h of cell efflux) of radioiodine ((125)I) were observed in hNIS and hTPO co-expressed PC-3 cells as compared to non-transfected PC-3 cells. We concluded that the new virus vector displayed favorable radioiodine uptake and retention properties in hNIS-hTPO transfected PC-3 cells. Our study will provide valuable information on improving the efficacy of hNIS-hTPO co-mediated radioiodine gene therapy.

Keywords: Gene therapy; gateway cloning system; hNIS; hTPO; prostate cance.

Figure 1

Schematic representation of the pAV.Ex1d-CMV>hNIS construction. The PCR products after hNIS amplification were used for a BP reaction, followed by bacterial transformation and sequencing verification. After the selection of positive clones, the pAV.Ex1d-CMV>hNIS gene was generated and validated by functional analysis.

Figure 2

Schematic representation of the pAV.Ex1d-CMV>hTPO/T2A/hNIS construction. The PCR products after hNISs and hTPOs amplification were used in the first step for a BP reaction, followed by bacterial transformation and sequencing verification. After the selection of positive clones, the pAV.Ex1d-CMV>hTPO/T2A/hNIS gene was generated and validated by functional analysis.

Figure 3

Characterization of hNIS and hTPO cDNA genes. A. Agarose gel electrophoresis of hNIS PCR products demonstrated a single band close to 2 K bp. B. Agarose gel electrophoresis of hTPO PCR products demonstrated a single band close to 7.2 K bp.

Figure 4

Characterization of attB1/hNIS/attB2 and attB1/hTPO/T2A/hNIS/attB2 by agarose gel electrophoresis. A. A single 2.0 K-band (Lane 1) confirmed the size of attB1/hNIS/attB2. B. Positive bands around 2.8 K bp (Lane 1) and 2.0 K bp (Lane 2) confirmed the sizes of attB1-hTPO/T2A and attB2-hNIS/T2A, respectively. C. A single 4.8 K-band (Lane 1) confirmed the size of attB1-hTPO/T2A/hNIS.

Figure 5

Characterization of pDown-hNIS and pDown-hTPO/T2A/hNIS. A. Agarose gel electrophoresis of pDown-hNIS PCR colony. A desired band with 2.2 K bp was shown in Lane 1–9. B. DNA sequence alignment of pDown-hNIS was found to be consistent with the hNIS sequence in the GenBank. C. Characterization of pDown-hTPO/T2A/hNIS after EcoRV and XhoI enzyme digestion. Positive bands with 2.6 K and 4.7 K bp were obtained which agreed with the desired bands at the positions of ~ 2.6 K and ~ 4.6 K bp. D. DNA sequence alignment of pDown-hTPO/T2A/hNIS was found to be consistent with the hTPO sequence in the GenBank.

Figure 6

Characterization of pAV.Ex1d-CMV>hNIS and pAV.Ex1d-CMV>hTPO/T2A/hNIS. A. Agarose gel electrophoresis of pAV.Ex1d-CMV>hNIS PCR colony. A desired band with 2.2 K bp was shown in Lane 1–7. B. DNA sequence alignment of pDown-hNIS was found to be consistent with the hNIS sequence in the GenBank. C. Characterization of pAV.Ex1d-CMV>hNIS after NdeI and BstBI enzyme digestion. A 4.7 K-band was observed which agreed with the desired size of 4.7 K bp. D. DNA sequence alignment of pAV.Ex1d-CMV>hTPO/T2A/hNIS was found to be consistent with the hTPO sequence in the GenBank.

Figure 7

Packaging of integrated Ad5 virus. CPE phenomena were observed in the transfected HEK293 cells (A. pAV.Ex1d-CMV>hNIS; B and C. pAV.Ex1d-CMV>hTPO/T2A/hNIS); but not observed in the non-transfected HEK293 cells (D). 1 × 10⁴ HEK293 cells/well; magnification 10× in A and B; magnification 20× in C and D.

Figure 8

Results of double-label immunofluorescence staining (magnification 20×). Stainings of hTPO (A. red color) and hNIS (B. green color) protein were displayed in pAV.Ex1d-CMV>hTPO/T2A/hNIS transfected PC-3 cells. Staining of hNIS protein (C. green color) was displayed in pAV. Ex1d-CMV>hNIS transfected PC-3 cells. No staining (D. red color field; E. green color field) was observed in the non-transfected PC-3 cells.

Figure 9

Time course of hTPO-hNIS-, hNIS-, and hTPO- mediated radioiodine (¹²⁵I) uptake (A) and retention (B) in transfected PC-3 cells as compared to non-transfected.