No abstract available.
Gastrointestinal Neoplasms* ; Thymidine Kinase* ; Thymidine*

Adenoviridae ; Humans ; Simplexvirus ; Thymidine Kinase

Transduction of retroviral-mediated herpes simplex virus thymidine kinase(HSVtk) gene into tumor cells and subsequent ganciclovir(GCV) treatment have been used as an experimental and clinical therapeutic strategy. Because non-transduced tumor cells can be killed by small proportion of transduced cells, known as bystander effect. Increasing bystander effect is useful strategy of suicidal gene therapy using HSVtk gene. To get a better bystander effect, we transduced T98G gliobastoma cells with HSVtk gene, single and double transduction with different marker genes respectively. Double HSVtk gene transduced cell lines showed significantly increased HSVtk activity(83%) by measuring the intracellular amount of phosphrylated 3H-GCV comparing to the single HSVtk gene transduced cell lines. In vitro bystander effect, examined by coculturing with HSVtk gene transduced cells and HSVtk- negative pa rental cells, was significantly increased on double HSVtk gene transduced cell lines. These results suggest that increasing herpes simplex virus thymidine kinase activity by double transfer of HSVtk gene into tumor cells can be a useful strategy for treating cancer with suicidal gene therapy.
Bystander Effect* ; Cell Line ; Ganciclovir ; Genetic Therapy ; Simplexvirus ; Thymidine Kinase* ; Thymidine*

OBJECTIVETo evaluate the method of adenovirus vector-mediated herpes simplex virus-thymidine kinase gene (ADV-TK)/ganciclovir(GCV) system and photodynamic therapy(PDT) for treating the oral malignant tumor.

METHODSTen patients who were suffering from oral malignant tumor of the different positions were selected, and injected with the hematoporphyrin derivative (HPD), irradiated by picking the corresponding laser frequency, and injected the ADV-TK gene into the tumor body and periphery. By the imaging and the hemodynamics analysis, the clinical efficacy after infusing vein with GCV was assessed.

RESULTSAfter the combination method treatments, patients' tumors appeared clear shrinkage or complete extinction. There was obvious fall of the blood flow volume in the tumor body. Imaging results showed significantly differences. So it was a perfect and effective treatment.

CONCLUSIONThere are many advantages to apply the ADV-TK/GCV system and PDT treatment on the oral malignant tumor, minimal side effects and greater clinical security. It is a safe and credible therapy which can be offered for curing the oral malignant tumor systematically.

Adenoviridae ; Ganciclovir ; Genetic Vectors ; Humans ; Neoplasms ; Photochemotherapy ; Simplexvirus ; Thymidine Kinase

OBJECTIVETo construct a replication-defective adenovirus containing TK gene and investigate the killing effects of TK gene against human liver cancer cells SMMC-7721.

METHODSThe recombinant adenovirus ADV-TK was constructed using homologous recombination in the cells. SMMC-7721 cells transfected with recombined adenovirus were exposed to GCV, and the cell viability was measured by MTT assays.

RESULTSThe recombinant adenovirus containing TK gene was successfully constructed. Transfection by the recombinant adenovirus ADV-TK and GCV exposure significantly suppressed the growth of SMMC-7721 cells.

CONCLUSIONA replication-defective adenovirus containing TK gene has been successfully constructed, and in combination with GCV, the recombinant adenovirus produces significant killing effect against SMMC-7721 cells in vitro.

Adenoviridae ; genetics ; Cell Line, Tumor ; Genetic Therapy ; Genetic Vectors ; Humans ; Liver Neoplasms ; therapy ; Thymidine Kinase ; genetics

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.
Animals ; Carcinoma, Hepatocellular* ; Cell Count ; Gene Expression ; Genes, Reporter ; Genetic Therapy ; Guanine* ; Mice ; Suicide ; Thymidine Kinase

BACKGROUND: Replication of defective adenoviral vectors can be used for gene transfer into a wide spectrum of replicating and nonreplicating cells. Accordingly, selective introduction of genes encoding for susceptibility to nontoxic drugs into proliferating tumor may be used to treat cancer. We investigated the efficacy of in vitro transduction of neuroblastoma cell with the herpes simplex virus thymidine kinase gene followed by administration of the antiviral drug ganciclovir. METHODS: The recombinant adenoviral vector ADV/TK carrying the HSV-TK gene, under the control of the promoter from Rous sarcoma virus long term terminal repeat was constructed. And 1 x 10(4) Neuro 2a cells were plated in 96 well cultured plates and infected with ADV/TK at multiplicity of infection of 0, 1, 10, and 100. Twenty-four hours later, the infected cells were treated with PBS or ganciclovir at a concentration of 10 g/ml. After 48hr, the surviving cells in 96 well plates were determined by MTT assay. RESULTS: After infection in vitro with ADV/TK at moi of 0, 1, 10, 100 and subsequent ganciclovir treatment, the percent survival rate of 1 x 10(4) Neuro 2a cells were 105%, 32%, 25%, and 15%. But the survival rate of 1 x 10(4) Neuro 2a cells with PBS treatment were 100%, 92%, 105%, 103%. CONCLUSION: We concluded that in vitro transduction of neuroblastoma cell with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir is very effective.
Cell Line* ; Ganciclovir* ; Neuroblastoma* ; Phosphotransferases ; Rous sarcoma virus ; Simplexvirus ; Survival Rate ; Terminal Repeat Sequences ; Thymidine Kinase

Knowledge of molecular mechanisms governing malignant transformation brings new opportunities for therapeutic intervention against cancer using novel approaches. One of them is gene therapy based on the transfer of genetic material to an organism with the aim of correcting a disease. The application of gene therapy to the cancer treatment has led to the development of new experimental approaches such as suicidal gene therapy, inhibition of oncogenes and restoration of tumor-suppressor genes. Suicidal gene therapy is based on the expression in tumor cells of a gene encoding an enzyme that converts a prodrug into a toxic product. Representative suicidal genes are Herpes simplex virus type 1 thymidine kinase (HSV1-tk) and cytosine deaminase (CD). Especially, physicians and scientists of nuclear medicine field take an interest in suicidal gene therapy because they can monitor the location and magnitude, and duration of expression of HSV1-tk and CD by PET scanner.
Cytosine Deaminase ; Genes, vif ; Genetic Therapy* ; Herpesvirus 1, Human ; Nuclear Medicine ; Oncogenes ; Thymidine Kinase

PURPOSE: Hepatocellular carcinoma (HCC) is one of the most common malignancy with high mortality in Korea. A new therapeutic modality such as gene therapy is necessary to improve the prognosis of hepatoma patients. Therefore we investigated the preclinical significance of Herpes simplex virus - thymidine kinase/ganciclovir (HSV-tk/GCV) gene therapy model using the retroviral vector for HCC cell lines. MATERIALS AND METHODS: LNC/HSV-tk retroviral vector and PA317/LNC/HSV-tk pro- ducer cell line were constructed. HSV-tk transduced HCC cells using the LNC/HSV-tk retrovirus were selected by the G418 containing media. In vitro GCV sensitivity test of the HCC cells was performed by MTT assay. To evaluate in vivo GCV sensitivity, GCV was intraperitoneally injected after subcutaneous administration of HCC cells into each flank of the nude mouse. RESULTS: HSV-tk gene transduction and expression in HCC cells were confirmed by RT-PCR. HSV-tk transduced HCC cell lines (SK-Hepl/HSV-tk and Hep-3B/HSV-tk) showed the marked GCV sensitivity comparing with the parental cell lines (SK-Hepl and Hep-3B) by MTT assay (p<0.001). The MTT test revealed that SK-Hepl/HSV-tk cells were more sensitive to GCV compare with that of Hep-3B/H5V-tk cells, and the parent cell line showed minimal growth suppression by the GCV treatment. In 12 nude mice received tumor cell mixtures of Hep-3B and Hep-3B/HSV-tk cells which contained more than 50% of HSV-tk transduced cells, the tumor was not developed in ll mice by the intraperitoneal administration of GCV. The tumors developed in 1 of 6 mice and 5 of 6 mice when mixtures contained 30% and 10% of HSV-tk transduced cells, respectively. Five mice out of 6 mice received inoculum containing the mixtures of 70% and 50% of HSV-tk transduced cells into each flank survived more than 6 month after HSV-tk/GCV treatment. Conelusion: HSV-tk gene transduced HCC cells showed the enhanced sensitivity to GCV. In nude mice HSV-tk/GCV strategy for HCC seemed to be more effective when tumor cell inoculum contained more than 30% of HSV-tk transduced HCC cells.
Animals ; Carcinoma, Hepatocellular* ; Cell Line* ; Ganciclovir* ; Genetic Therapy ; Herpes Simplex* ; Humans ; Korea ; Mice ; Mice, Nude ; Mortality ; Parents ; Prognosis ; Retroviridae* ; Simplexvirus* ; Thymidine Kinase* ; Thymidine* ; Zidovudine

Tumor cell proliferation is considered to be a useful prognostic indicator of tumor aggressiveness and tumor response to therapy, but in vitro measurement of individual proliferation is complex and tedious work. PET imaging provides a noninvasive approach to measure tumor growth rate in situ. Early approaches have used 18F-FDG or methionine to monitor proliferation status. These 2 tracers detect changes in glucose and amino acid metabolism, respectively, and therefore provide only an indirect measure of proliferation status. More recent studies have focused on DNA synthesis itself as a marker of cell proliferation. Cell lines and tissues with a high proliferation rate require high rates of DNA synthesis. [11C]Thymidine was the first radiotracer for noninvasive imaging of tumor proliferation. The short half-life of 11C and rapid metabolism of [11C]thymidine in vivo make the radiotracer less suitable for routine use. Halogenated thymidine analogs such as 5-iodo-2-deoxyuridine (IUdR) can be successfully used as cell proliferation markers for in vitro studies because these compounds are rapidly incorporated into newly synthesized DNA. IUdR has been evaluated as a potential in vivo tracer in nuclear medicine, but the image quality and the calculation of proliferation rates are impaired by its rapid in vivo degradation. Hence, the thymidine analog 3'-deoxy-3'-18F-fluorothymidine (FLT) was recently introduced as a stable proliferation marker with a suitable nuclide half-life and stable in vivo. [18F]FLT is phosphorylated to 3-fluorothymidine monophosphate by thymidine kinase 1 and reflects thymidine kinase 1 activity in proliferating cell. [18F]FLT PET is feasible in clincal use and well correlates with cellular proliferation. Choline is a precursor for the biosynthesis of phospholipids (in particular, phosphatidylcholine), which is the essential component of all eukaryotic cell membranes and [11C]choline, which is a new marker for cellular proliferation.
Cell Line ; Cell Proliferation* ; Choline ; DNA ; Eukaryotic Cells ; Fluorodeoxyglucose F18 ; Glucose ; Half-Life ; Idoxuridine ; Membranes ; Metabolism ; Methionine ; Nuclear Medicine ; Phospholipids ; Thymidine ; Thymidine Kinase