ABSTRACT
PURPOSE: Hydrodynamic-based procedure is a simple and effective gene delivery method to lead a high gene expression in liver tissue. Non-invasive imaging reporter gene system has been used widely with herpes simplex virus type 1 thymidine kinase (HSV1-tk) and its various substrates. In the present study, we investigated to image the expression of HSV1-tk gene with 5-(2-iodovinyl)-2'-deoxyuridine (IVDU) in mouse liver by the hydrodynamicbased procedure. MATERIALS AND METHODS: HSV1-tk or enhanced green fluorescence protein (EGFP) encoded plasmid DNA was transferred into the mouse liver by hydrodynaminc injection. At 24 h post-injection, RT-PCR, biodistribution, fluorescence imaging, nuclear imaging and digital wholebody autoradiography (DWBA) were performed to confirm transferred gene expression. RESULTS: In RT-PCR assay using mRNA from the mouse liver, specific bands of HSV1-tk and EGFP gene were observed in HSV1-tk and EGFP expressing plasmid injected mouse, respectively. Higher uptake of radiolabeled IVDU was exhibited in liver of HSV1-tk gene transferred mouse by biodistribution study. In fluorescence imaging, the liver showed specific fluorescence signal in EGFP gene transferred mouse. Gamma-camera image and DWBA results showed that radiolabeled IVDU was accumulated in the liver of HSV1-tk gene transferred mouse. CONCLUSION: In this study, hydrodynamic-based procedure was effective in liver-specific gene delivery and it could be quantified with molecular imaging methods. Therefore, co-expression of HSV1-tk reporter gene and target gene by hydrodynamic-based procedure is expected to be a useful method for the evaluation of the target gene expression level with radiolabeled IVDU.
Subject(s)
Animals , Mice , Autoradiography , DNA , Fluorescence , Gene Expression , Genes, Reporter , Herpes Simplex , Herpesvirus 1, Human , Idoxuridine , Liver , Methylmethacrylates , Molecular Imaging , Optical Imaging , Plasmids , Polystyrenes , RNA, Messenger , Simplexvirus , Thymidine KinaseABSTRACT
PURPOSE: The HSV1-tk gene has been extensively studied as a type of reporter gene. In hepatocellular carcinoma (HCC), only a small proportion of patients are eligible for surgical resection and there is limitation in palliative options. Therefore, there is a need for the develoopement of new treatment modalities and gene therapy is a leading candidate. In the present study, we investigated the usefulness of substrate, 2'-fluoro-2'-deoxy-1-beta-D-arabino-furanosyl-5-[124/125I]iodo- uracil ([124/125I]FIAU) as a non-invasive imaging agent for HSV1-tk gene therapy in hepatoma model using small animal PET. MATERIAL AND METHODS: With the Morris hepatoma MCA cell line and MCA-tk cell line which was transduced with the HSV1-tk gene, in vitro uptake and correlation study between [125I]FIAU uptake according to increasing numeric count of percentage of MCA-tk cell were performed. The biodistribution data and small animal PET images with [124I]FIAU were obtained with Balb/c-nude mice bearing both MCA and MCA-tk tumors. RESULTS: Specific accumulation of [125I]FIAU was observed in MCA-tk cells but uptake was low in MCA cells. Uptake in MCA-tk cells was 15 times higher than that of MCA cells at 480 min. [125I]FIAU uptake was linearly correlated (R2=0.964, p=0.01) with increasing percentage of MCA-tk numeric cell count. Biodistribution results showed that [125I]FIAU was mainly excreted via the renal system in the early phase. Ratios of MCA-tk tumor to blood acting were 10, 41, and 641 at 1 h, 4 h, and 24 h post-injection, respectively. The maximum ratio of MCA-tk to MCA tumor was 192.7 at 24 h. Ratios of MCA-tk tumor to liver were 13.8, 66.8, and 588.3 at 1 h, 4 h, and 24 h, respectively. On small aninal PET, [124I]FIAU accumulated in substantial higher levels in MCA-tk tumor and liver than MCA tumor. CONCLUSION: FIAU shows selective accumulation to HSV1-tk expressing hepatoma cell tumors with minimal uptake in normal liver. Therefore, radiolabelled FIAU is expected to be a useful substrate for non-invasive imaging of HSV1-tk gene therapy and therapeutic response monitoring of HCC.
Subject(s)
Animals , Humans , Mice , Arabinofuranosyluracil , Carcinoma, Hepatocellular , Cell Count , Cell Line , Genes, Reporter , Genetic Therapy , Herpes Simplex , Herpesvirus 1, Human , Liver , Liver Neoplasms, Experimental , Methylmethacrylates , Polystyrenes , Simplexvirus , Statistics as Topic , Uracil , UrsidaeABSTRACT
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
Objective To evaluate the therapeutic effect and the mechanism of herpes simplex virus thymidine kinase Ⅰ type (HSV 1\|tk) and Ganciclovir(GCV) in animal model of human glioma. Methods We demonstrated that tk gene was stably integrated into the genome of retrovirus vector producing cell line pN 2A\|tk/VPC(VPC) by Southern blot hybridization; then we established the nude mice model of glioma by inoculating the VPC and the human glioma cells DBTRG\|05MG(05MG) at 1∶1,1∶4 ratios respectively(only inoculated 05MG cells as control group), then GCV treatment was administrated i.p.,30mg/(kg\5d), twice daily for 14 days. A week later, the nude mice were sacrificed and the pathological sections of tumor tissues were observed by light microscopy. Results The tumor mass of 1∶1 group decreased remarkably compared with 1∶4 group or control group respectively ( P