ABSTRACT
PURPOSE: The HSV1-tk reporter gene system is the most widely used system because of its advantage that direct monitoring is possible without the introduction of a separate reporter gene in case of HSV1-tk suicide gene therapy. In this study, we investigate the usefulness of the reporter probe (substrate), 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) for non-invasive reporter gene imaging using PET in HSV1-tk expressing hepatoma model. MATERIALS AND METHODS: Radiolabeled FHBG was prepared in 8 steps from a commercially available triester. The labeling reaction was carried out by NCA nucleophilic substitution with K[18F]/K2.2.2. in acetonitrile using N2-monomethoxytrityl-9-[4-(tosyl)-3-monomethoxytritylmethylbutyl]guanine as a precursor, followed by deprotection with 1 N HCl. Preliminary biological properties of the probe were evaluated with MCA cells and MCA-tk cells transduced with HSV1-tk reporter gene. In vitro uptake and release-out studies of [18F]FHBG were performed, and was analyzed correlation between [18F]FHBG uptake ratio according to increasing numeric count of MCA-tk cells and degree of gene expression. MicroPET scan image was obtained with MCA and MCA-tk tumor bearing Balb/c-nude mouse model. RESULTS: [18F]FHBG was purified by reverse phase semi-HPLC system and collected at around 16-18 min. Radiochemical yield was about 20-25% (corrected for decay), radiochemical purity was >95% and specific activity was around >55.5 GBq/micro mol. Specific accumulation of [18F]FHBG was observed in HSV1-tk gene transduced MCA-tk cells but not in MCA cells, and consecutive 1 hour release-out results showed more than 86% of uptaked [18F]FHBG was retained inside of cells. The uptake of [18F]FHBG was showed a highly significant linear correlation (R2=0.995) with increasing percentage of MCA-tk numeric cell count. In microPET scan images, remarkable difference of accumulation was observed for the two type of tumors. CONCLUSION: [18F]FHBG appears to be a useful as non-invasive PET imaging substrate in HSV1-tk expressing hepatoma model.
Subject(s)
Animals , Mice , Carcinoma, Hepatocellular , Cell Count , Gene Expression , Genes, Reporter , Genetic Therapy , Guanine , Suicide , Thymidine KinaseABSTRACT
PURPOSE: The herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) is an attractive candidate as a reporter gene in noninvasive reporter gene monitoring system. The HSV1-tk gene was chosen as a reporter gene, because it has been extensively studied, and there are appropriate reporter probes, substrates of HSV1-tk gene product, to apply for HSV1-tk gene imaging. We used radiolabeled 5-iodovinyl-2'-deoxyuridine (IVDU) and 5-Iodovinyl-2'-fluoro-2'-deoxyuridine (IVFRU) as reporter probes for HSV1-tk gene monitoring system. MATERIALS AND METHODS: We prepared HSV1-tk gene transduced Morris hepatoma cell line using retroviral vector, MOLTEN containing HSV1-tk gene. And we confirmed the HSV1-tk gene expression by Northern blotting and Western blotting. We compared in vitro uptakes of radioiodinated IVDU and IVFRU to monitor HSV1-tk gene expression in Morris hepatoma cell line (MCA) and HSV1-tk gene tranduced MCA (MCA-tk) cells until 480 minutes. We also performed correlation analysis between percentage of HSV1-tk gene tranduced MCA cell % (MCA-tk%) and uptakes of radiolabeled IVDU or IVFRU. RESULTS: MCA-tk cell expressed HSV1-tk mRNA and HSV1-TK protein. Two compounds showed minimal uptake in MCA, but increased uptake was observed in MCA-tk. IVDU showed 4-fold higher accumulation than IVFRU at 480 min in MCA-tk (p 0.96) with increasing MCA-tk%. CONCLUSION: The radiolabeld IVDU and IVFRU showed higher specific accumulation in retrovirally HSV1-tk gene transfected Morris hepatoma cell line. Both IVDU and IVFRU could be used as good substrates for evaluation of HSV1-tk gene expression.
Subject(s)
Animals , Blotting, Northern , Blotting, Western , Cell Line , Gene Expression , Genes, Reporter , Genetic Therapy , Herpes Simplex , Herpesvirus 1, Human , Liver Neoplasms, Experimental , RNA, Messenger , Simplexvirus , Thymidine Kinase , Thymidine , ZidovudineABSTRACT
No abstract available.
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ABSTRACT
This study was designed to prospect the 'In-labelled paclitaxel as tumor imaging agent. In order to provide a taxol molecule with a functional group which is able to chelate In-lll, taxol-DTPA conjugate and 2-hemisuccinyltaxol were synthesized by esterification of taxol at C-2 on C-13 carbon with DTPA anhydride and succinic anhydride, respectively. Synthesis yield of the taxol derivatives was 34% for taxol- DTPA and 80% for 2'-hemisuccinyltaxol. Cytotoxicity of the taxol derivatives were measured by MTT method toward cell lines HT29, B16, P388, and CT26. The cytotoxic activities of the taxol derivatives were maintained, although less active than taxol. Radiolabelling of the taxol derivatives were proceeded directly with 111InCh or indirectly with 111In-citrate(ligand-exchange method). The ligand-exchane methocl was not suitable because some precipitat:es appeared during the reaction. On the contrary, by direct radiolabelling methnd, we were able to obtain taxol DTPA-111In in 100% radiochemical yield. However, 2'-hemisuccinyltaxol was not labellecl by both methods. Yield and radiochemiral purity of the radiolabelled com- pound were determined by HPI.C, paper chromatography and instant thin layer chromatography. Taxol-DTPA-111In was characterized to be hydrophilic by lipophi- licity test, and nearly non-adhesive to HT29, E316, P388, and CT26 by cell hinding affinity test. Binding affinity of the taxol-DTPA-111In complex to serum proteins was also examined by protein precipitation with 30% trichloroacetic acid. The results showed that 309o of the taxol-DTPA-111In complex binds with serum proteins.
Subject(s)
Blood Proteins , Carbon , Cell Line , Chromatography, Paper , Chromatography, Thin Layer , Esterification , Paclitaxel , Pentetic Acid , Trichloroacetic AcidABSTRACT
No abstract available.