No abstract available.
Thymidine

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

BACKGROUND: Many types of cancer become resistant to current chemotherapeutic and radiotherapeutic intervention. To overcome this situation application of gene therapy by the introduction of suicide genes followed by their prodrugs may be promising. A viral enzyme, Herpes simplex thymidine kinase (HSV-tk), which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining prodrug-activating gene therapy and irradiation might result in enhanced antitumor effects. METHODS: The HSV-tk gene was cloned into the retroviral vector, pLXSN and established the clones producing retroviruses carrying the HSV-tk gene. The carcinoma cell line, HCT116 and Huh-7 were transduced with high-titer recombinant retroviruses. These cell lines were treated with ganciclovir before or after irradiation for the defining combinational effect of suicide gene therapy and radiotherapy. RESULTS: The titers of cloned PA3 17 amphotropic retroviruses ranged from 4 to 6 X 10(6) CFU/ml . After selectional periods, the expression of HSV-tk was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). The growth of cells expressing HSV-tk was inhibited as increase of GCV dose after 48 hr and the growth inhibitory effect of GCV was much higher after 72 hr. When the cells transduced with HSV-tk gene were exposed to radiation, the growth inhibitory effect of GCV was significantly increased, as compared with non-transduced parental cells. CONCLUSIONS: The result s suggest that the addition of HSV-tk gene therapy to standard radiation therapy may improve the effectiveness of treatment for solid tumors.
Cell Line ; Clone Cells ; Ganciclovir* ; Genetic Therapy ; Herpes Simplex* ; Humans ; Parents ; Phosphorylation ; Polymerase Chain Reaction ; Prodrugs ; Radiotherapy ; Retroviridae ; Simplexvirus* ; Suicide ; Thymidine Kinase* ; Thymidine* ; Zidovudine

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< 0.01). Both IVDU and IVFRU uptake were linearly correlated (R2> 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.
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* ; Zidovudine

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

BACKGROUND: Metabolic cooperation via gap junctional intercellular communication (GJIC) is an important mechanism of the bystander effect in gene therapy using the Herpes Simplex Virus thymidine kinase/ganciclovir (HSVtk) 'prodrug' system. Since retinoids have been reported to increase GJIC by induction of connexin 43 expression, we hypothesized that treatment of tumor cells with retinoic acid could augment the bystander erect of the HSVtk/GCV system and result in improved tumor cell killing by enhancing GJIC. METHODS: We transferred HSVtk gene to SKHep-J cell line that does not express connexin43, and also transferred the gone to human and murine mesothelioma cell lines that express connexin43. We verified that retinoic acid enhanced GJIC utilizing a functional double-dye transfer study and evaluated the effects of retinoic acid on the growth rate of honor cells. We then tested the effects of retinoic acid on bystander-mediated cell killing. RESULTS: Addition of all-trans retinoic acid (RA) increased GJIC in cell lines expressing connexin43 and was associated with more efficient in vitro bystander killing in cells transduced with HSVtk via adenoviral and retroviral vectors. In contrast, there was no increase in the efficiency of the bystander effect after exposure to RA in a cell line which had no detectable connexin 43. CONCLUSION: These results provide evidence that retinoids can augment the efficiency of cell killing with the HSVtk/GCV system by enhancing bystander effect and may thus be a promising new approach to improve responses in gene therapy utilizing the HSVtk system to treat tumors.
Bystander Effect* ; Cell Line ; Connexin 43 ; Genetic Therapy* ; Herpes Simplex* ; Homicide ; Humans ; Mesothelioma ; Phosphotransferases* ; Retinoids ; Simplexvirus* ; Thymidine ; Tretinoin* ; Zidovudine

PURPOSE: Gastric cancer is the most common malignancy in Korea. Although treatment such as surgery, chemotherapy, and immunotherapy has greatly improved, the mortality rate of gastic cancer is still high, A new therapeutic trial is necessary to improve the cure rate of gastric cancer. Therefore we investigated the pre-clinical significance of HSV-tk gene therapy using retroviral vector for gastric cancer cell lines. MATERIALS AND METHODS: LNC/HSV-tk retroviral vector and PA317/LNC/HSV-tk producer cell line were constructed. HSV-tk gene transduction and expression were detected by PCR. An in vitro ganciclovir(GCV) sensitivity test was performed by MTT assay. To evaluate in vivo GCV sensitivity, GCV was intraperitoneally injected after tumor formation in the nude mice. Bystander effect was observed in vitro MTT assay using YCC- S-2 cell line and in vivo using N87 and YCC-S-2 cell lines. RESULTS: The in vitro GCV sensitivity test showed that the growth inhibition was 30~32% with 0.5 uM GCV and 52~77% with 500 uM GCV in the HSV-tk transduced cell line in comparison with 0- 5% with 0.5 and 500 uM GCV in the parent cell line. The in vivo GCV administration showed that the tumors induced by HSV-tk transduced N87 cell line and YCC-S-2 cell line decreased completely, while the tumors with the parent cell lines continued to grow in nude mice. We observed no tumor cells in tissue specimen of the tumor induced by the N87/HSV-tk cell line after. GCV administration. In vitro and in vivo bystander effects were observed in HSV-tk/GCV system due to the resultant cell death exceeding the proportion of HSV-tk transduced cells in the mixtures of HSV-tk transduced and parent cells. CONCLUSION: HSV-tk transduced gastric cancer cell lines showed sensitivity to GCV and a bystander effect was observed. These results suggested that HSV-tk/GCV system should be evaluated in the clinical settings.
Animals ; Bystander Effect ; Cell Death ; Cell Line* ; Drug Therapy ; Ganciclovir* ; Genetic Therapy ; Herpes Simplex* ; Humans ; Immunotherapy ; Korea ; Mice ; Mice, Nude ; Mortality ; Parents ; Polymerase Chain Reaction ; Stomach Neoplasms* ; Thymidine Kinase* ; Thymidine* ; Zidovudine

OBJECTIVE: The bystander effect, the phenomenon that non-transduced tumor cells can be killed along with the transduced cells, enables suicidal gene therapy feasible in spite of low efficiency of gene transfer at present time. Increment of thymidine kinase activity in the cells through double copy insertion of HSVtk gene may lead to increased bystander effect in vitro and in vivo, therefore enhancing the therapeutic potential of suicidal gene therapy. To examnine this hypothesis, we did an experiment to improve bystander effect by using double transfer of HSVtk gene into 9L tuomr cells. METHODS: We transduced 9L glioblastoma cells which had one copy or two copies of HSVtk gene using retroviral vector. Two different retroviral vector plasmids containing HSVtk gene were made employing pBabePuro or LXSN plasmid whose selection markers were puromycin and G418, respectively(LtkSP and LtkSN). Recombinant retrovirus was produced from ecotrophic PA317 packaging cells. Infection of the 9L cells with LtkSP recombinant retrovirus and selection with puromycin was done in vitro to make 9L/LtkSP(9LtkS). These cells were infected again with LtkSN recombinant retrovirus and selected under G418 to establish cells containing two copies of HSVtk gene, 9L/LtkSP/ LtkSN(9LtkD). RESULTS: By measuring the intracellular amount of phosphorylated 3H-GCV, 9LtkD cell lines showed significantly increased HSVtk activity, being 70% higher than that of 9LtkS cell lines. The sensitivity to GCV was also markedly increased. In vitro bystander effect, examined by coculturing with HSVtk gene transduced cells and 9L cells, was significantly increased on 9LtkD cell lines. To evaluate the in vivo bystander effect, 9LtkD/9L(20%:80%) cells or 9LtkS/9L (20%:80%) cells were implanted into the brains of Fisher 344 inbred rats, followed by administration of ganciclovir. Rats implanted with 9LtkD/9L cells showed better tumor regression and longer survival than those of 9LtkS/9L after treatment with ganciclovir. CONCLUSION: These results suggest that increasing herpes simplex virus thymidine kinase activity by introducing double copy of HSVtk gene into tumor cells would improve in vitro and in vivo bystander effect, and lead to enhanced efficacy of suicidal gene therapy.
Animals ; Brain ; Bystander Effect* ; Cell Line ; Ganciclovir ; Genetic Therapy ; Glioblastoma ; Glioma ; Herpes Simplex* ; Phosphotransferases* ; Plasmids ; Product Packaging ; Puromycin ; Rats ; Retroviridae ; Simplexvirus* ; Thymidine Kinase ; Zidovudine

BACKGROUND: The antitumor effects of herpes simplex virus thymidine kinase(HSV-tk) and ganciclovir(GCV) strategies for cancer gene therapy have a the following advantages:1) a direct cytotoxicity to HSV-tk modified cancer cells by GCV 2) a cell death by the local transfer of toxic metabolites from the HSV-tk modified cells to nearby unmodified tumor cells(bystander effect), and 3) in vivo bystander effect such as antitumor-immunity. Retroviral and adenoviral sequences can silence transgene expression in cells and mice. In this study, we investigated the above described advantages of HXV-tk/GCV strategy in Lewis lung cell and mouse lung cancer model using retroviral vector and adenoviral vector. Also, we observed whether the expression of a silenced gene can be reactivated by treating cell with butyrate. METHODS: Retrovirus-HSV-tk and adenovirus-HSV-tk vectors were used for the transduction of Lewis lung carcinoma(LLC) cells. The change of HSV-tk expression by butyrate was measured by Western blot.The antitumor activities containing bystander effect were observed in vivo(by MTT assay) and in vivo tumor models of various combinations of LLC and LLC-tk. RESULTS: 1. Butyrate induced the enhancement of HSV-tk expression from adenovirally transduced cells but not from retrovirally transduced cells. 2. Both retrovirus-HSV-tk and adenovirus-HSV-tk vectors with GCV treatment were effective for killing of tumor cell in vitro and suppression of LLC tumorigenicity. Bystander effect was responsible for killing of mixture of LLC-tk and LLC in vitro and in vivo-tumorigenicity model. CONCLUSION: Butyrate could augment adenoviral vector seems to be an effective approach for lung cancer therapy.
Adenoviridae ; Animals ; Butyrates ; Bystander Effect ; Carcinoma, Lewis Lung* ; Cell Death ; Genes, Neoplasm ; Genetic Therapy* ; Herpes Simplex* ; Homicide ; Lung ; Lung Neoplasms ; Mice* ; Phosphotransferases* ; Retroviridae ; Simplexvirus* ; Thymidine ; Transgenes ; Zidovudine*

BACKGROUND: Transplantation of cardiac myocytes (CMs) into the injured heart emerges as a potential alternative for the treatment of heart failure. Genetic modification of CMs could enhance and/or modify its therapeutic effects. The characteristics of retroviral gene delivery, which is most commonly used in human trial, has been minimally studied in CMs due to its low efficiency in non-dividing cells. In this study, using newly developed high-titer retrovirus, we evaluated 1) the efficiency of gene transfer into CMs, 2) whether S phase during infection is necessary for the transduction, and 3) characteristics of gene delivery to mononucleated vs binucleated CMs. METHODS: Enriched CMs were cultured from the ventricles of 1 day-old rat hearts. The cells were transduced by MFG-nls-LacZ retroviruses (5x107 IU/ml) in the presence or absence of polybrene. 3H-thymidine was added to label cells in S phase. The cells were stained for b-galactosidase activity and then immunostained using MF20Ab to identify CMs. The cells were subsequently processed for in vitro autoradiography. RESULTS: 1)With 3 rounds of infection, 5.9% of total cultured cells were LacZ-positive. The efficiency of transduction reached upto 7.4% in the presence of polybrene 8 microgram/ml. 2)Nuclear morphology of LacZ-positive CMs was pleomorphic from mononucleated to multinucleated, mostly binucleated. 3)Among mononucleated CMs, 17% of cells were labelled with thymidine. Transduction efficiency (TDE) of thymidine-positive and -negative mononucleated CMs were 37.9% and 3.1%, respectively. Among binucleated cells, 28.9% of cells were labelled with thymidine. TDE of thymidine-positive and -negative binucleated CMs were 75.4% and 13.6%, respectively. 4)In total, TDE of binucleated cells were 3.5 times compared to the one of mononucleated cells (31.5% vs 9.0%). CONCLUSION: TDE of CMs using high-titer retrovirus is relatively low. S phase of cells during retroviral infection is not mandatory for the retroviral transduction. Binucleated CMs are susceptible to retroviral gene delivery and their TDE is higher than the one of mononucleated CMs.
Animals ; Autoradiography ; Cells, Cultured ; Dichlorodiphenyldichloroethane ; Genetic Therapy ; Heart ; Heart Failure ; Hexadimethrine Bromide ; Humans ; Myocytes, Cardiac* ; Rats ; Retroviridae* ; S Phase ; Thymidine ; Zidovudine