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Generation of Destabilized Herpes Simplex Virus Type 1 Thymidine Kinase as Transcription Reporter for PET Reporter Systems in Molecular–Genetic Imaging

¹ Institute of Medical Science, China Medical University, Taichung, Taiwan; ² Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan; ³ Institute of Radiological Sciences, Central Taiwan University of Science and Technology, Taichung, Taiwan; ⁴ National PET/Cyclotron Center, Taipei Veterans General Hospital, Taipei, Taiwan; ⁵ Department of Experimental Diagnostic Imaging, University of Texas M.D. Anderson Cancer Center, Houston, Texas; and ⁶ Department of Nuclear Medicine, Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan

View larger version (27K): [in this window] [in a new window]   FIGURE 1.  (A) Fluorescent photomicrographs of NG4TL4 cells expressing native HSV1-TK:GFP. (B) Fluorescent photomicrographs of NG4TL4 cells expressing mNLSHSV1-TK:GFP with mutations in N-terminal NLS. (C) 8-3H-ganciclovir accumulation in NG4TL4 control cells, HSV1-TK:GFP–transfected NG4TL4 cells, and mNLSHSV1-TK:GFP–transfected NG4TL4 cells. Bars represent mean ± SD of 3 independent experiments.

View larger version (29K): [in this window] [in a new window]   FIGURE 2.  (A) Western blot analysis of protein stabilities of mNLSHSV1-TK and mNLSHSV1-TK:dMODC in living NG4TL4 cells, with or without treatment with 0.1 mg of cycloheximide per milliliter. Cell lysates were subjected to sodium dodecyl sulfate (SDS) gel electrophoresis and transferred onto membrane. mNLSHSV1-TK and mNLSHSV1-TK:dMODC were detected with monoclonal antibody against TK. (B) Stability of mNLSHSV1-TK and mNLSHSV1-TK:dMODC in living NG4TL4 cells, with or without treatment with 0.1 mg of cycloheximide per milliliter. HSV1-TK activity of lysed cells was determined by in vitro HSV1-TK enzyme activity assay. Bars represent mean ± SD of 3 independent experiments.

View larger version (20K): [in this window] [in a new window]   FIGURE 3.  (A) Fluorescence stabilities of HSV1-TK:GFP and mNLSHSV1-TK:dMODC:GFP in presence of cycloheximide, and with or without MG-132 (26S proteasome inhibitor), were examined with fluorescence microscope. (B) TK enzyme stabilities of HSV1-TK:GFP and mNLSHSV1-TK:dMODC:GFP in presence of cycloheximide, and with or without MG-132 (26S proteasome inhibitor), were measured by HSV1-TK enzyme activity assay. NG4TL4 cells were transfected with vectors expressing these 2 proteins. After 24 h, transfected cells were treated with 100 mg of cycloheximide per milliliter for 6 h, with or without 40 µM MG-132. Fluorescence stabilities and TK enzyme stability of these fusion proteins were examined with fluorescence microscope and HSV1-TK enzyme activity assay, respectively.

View larger version (13K): [in this window] [in a new window]   FIGURE 4.  In vitro cell growth of NG4TL4 control cells, HSV1-TK–transfected NG4TL4 cells, mNLSHSV1-TK–transfected NG4TL4 cells, and mNLSHSV1-TK:dMODC–transfected NG4TL4 cells. Cell growth was analyzed using trypan blue staining assay. Data points represent mean ± SD of 3 independent experiments.

View larger version (22K): [in this window] [in a new window]   FIGURE 5.  (A) Doxycycline-mediated inhibition of HSV1-TK messenger RNA for NG4TL4/tet-off-HSV1-TK cells, NG4TL4/tet-off-mNLSHSV1-TK cells, and NG4TL4/tet-off-mNLSHSV1-TK:dMODC cells. (B) Doxycycline-mediated inhibition of HSV1-TK enzyme activity for NG4TL4/tet-off-HSV1-TK cells, NG4TL4/tet-off-mNLSHSV1-TK cells, and NG4TL4/tet-off-mNLSHSV1-TK:dMODC cells. Stably transformed cell lines were treated with 0, 0.01, 0.1, 1, and 10 ng of doxycycline per milliliter. After doxycycline treatment, changes in messenger RNA levels and enzyme activity of HSV1-TK were assayed at various time points using real-time PCR and in vitro assay for HSV1-TK enzyme activity, respectively. Bars represent mean ± SD of 3 independent experiments.

View larger version (14K): [in this window] [in a new window]   FIGURE 6.  Dynamic gene expression of HSV1-TK, mNLSHSV1-TK, or mNLSHSV1-TK:dMODC mediated by series of doxycycline inductions. HSV1-TK, mNLSHSV1-TK, and mNLSHSV1-TK:dMODC gene expression of NG4TL4/tet-off-HSV1-TK cells, NG4TL4/tet-off-mNLSHSV1-TK, and NG4TL4/tet-off-mNLSHSV1-TK:dMODC cell lines mediated by series of doxycycline inductions were monitored using in vitro assay for HSV1-TK enzyme activity. Open and closed arrows indicate changes in medium with or without doxycycline, respectively. Data points represent mean ± SD of 3 independent experiments.

View larger version (41K): [in this window] [in a new window]   FIGURE 7.  Sequential PET studies of tumors growing in FVB/N female mice. NG4TL4/tet-off-mNLSHSV1-TK:dMODC cells (dTK), G4TL4/tet-off-mNLSHSV1-TK cells (mNLSTK), NG4TL4/tet-off-HSV1-TK cells (TK), or NG4TL4 control cells (wild-type [WT]) were injected subcutaneously into 3 separate sites in single mice. When tumors reached at least 5 mm, mouse was imaged with 18F-FHBG on sequential days. Doxycycline (500 µg/mL) was administered intraperitoneally and also added to cage water supply for 1 d. Mouse was scanned with 18F-FHBG again 24 h after exposure to doxycycline.