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*

PURPOSE: To demonstrate the bystander effect in murine neuroblastoma model which transduced with HSV-TK gene in vitro and in vivo. METHODS: The LNC/TK vector was transfered in vitro into the neuro-2a cells, murine neuroblastoma cell line. Variable mixed populations of neuro-2a cells consisting of HSV-TK+ or HSV-TK- were plated into culture plates and treated with GCV for another 4 days. Surviving cells were counted and cell viability was determinated. For investigating the in vivo bystander effect, variable mixed populations of neuro-2a cells consisting of HSV-TK+ and HSV-TK- were inoculated into A/J mice. The tumor size was measured following injection of GCV for 7 days. RESULTS: The survival rate of the 100% neuro-2a/TK group was 90%, 25%, 5% and 0%, of 50% neuro-2a/TK group was 92%, 30%, 10% and 0%, and of the 10% neuro-2a/TK group was 95%, 40%, 15% and 5%. But, the survival rate of 0% neuro-2a/TK group was 120%, 150%, 180% and 220% on days 1, 2, 3, and 4 respectively. In the 100% and 50% neuro-2a/TK groups, tumor had disappeared following administration of GCV and in 10% neuro-2a/TK group, tumor size was not increased during GCV treatment. In 0% neuro-2a/TK group, tumor size increased during administration of GCV and all mice died after 6 weeks. CONCLUSIONS: We demonstrated the bystander effect in a murine neuroblastoma model which transduced with HSV-TK gene in vitro and in vivo. These results suggest that HSV-TK/GCV gene therapy may be useful for treatment of neuroblastoma.
Animals ; Bystander Effect* ; Cell Line ; Cell Survival ; Genetic Therapy* ; Mice ; Neuroblastoma* ; Survival Rate

No abstract available.
Bystander Effect* ; Carcinoma, Hepatocellular* ; Cell Line* ; Ganciclovir* ; Herpes Simplex* ; Phosphotransferases* ; Simplexvirus*

PURPOSE: Gap junction intercellular communication(GJIC) is an important mechanism of the bystander effect in herpes simplex thymidine kinase/ganciclovir(HSVtk/GCV) gene therapy Therefore, we attempted to enhance the bystander effect in vitro by exogenous overexpressing connexin 37(Cx37) in cells to increase GJIC. METHODS: NIH3T3 cells were transfected with the Cx37 and HSVtk gene or the HSVtk gene alone by the calcium phosphate method, and we detected their expression from these cells by RT-PCR. GCV-mediated cytotoxicity and the bystander effect of each transfectant was then assessed and compared. RESULTS: Cells transfected with HSVtk became sensitive to low concentration of GCV. We found significantly increased cytotoxicity in HSVtk/GCV gene therapy after introduction of the HSVtk and Cx37 genes together compared with the cytotoxicity seen after introduction of the HSVtk gene in vitro. Co-expression of the HSVtk and Cx37 genes potentiates HSVtk/GCV gene therapy through the bystander effect. CONCLUSION: These results indicated that the increase of GJIC using Cx37 have potentiated the by stander effect of HSVtk/GCV therapy, and may be a new approach to improve response in suicidal cancer gene therapy.
Bystander Effect* ; Calcium ; Gap Junctions* ; Genes, Neoplasm ; Genetic Therapy* ; Herpes Simplex ; Thymidine

PURPOSE: Selective introduction of genes conferring chemosensitivity into proliferating tumor cells may be used to treat cancer. We first investigated the bystander effect of retrovirus-mediated gene transfer of herpes simplex virus thymidine kinase(HSV-TK) gene to murine neuroblstoma cell line(neuro-2a) in vitro and in vivo. Second, we examined the mechanism and its enhancement of the bystander effect in murine neuroblastoma. METHODS: To investigate the bystander effect, we studied tumor growth and survival time after HSV-TK/ganciclovir(GCV) treatment in a syngenic A/J mouse neuroblastoma model by mixing various ratios of HSV-TK-expressing neuro-2a cells with wild type neuro-2a cells followed by GCV treatment. To investigate the mechanism of the bystander effect in murine neuroblastoma, immunohistochemistry using connexin 43, CD4 and CD8-specific monoclonal antibodies was analyzed. We studied whether IL-2-secreting neuro-2a cells(neuro-2a/IL-2) would potentiate the bystander effect. RESULTS: A strong bystander effect was observed in vitro and in vivo. The bystander effect in murine neuroblastoma was dependent on the immune response rather than connexin-mediated gap junction. Neuro-2a/IL-2 treatment enhanced the bystander effect in the HSV-TK/GCV system in murine neuroblastoma model. CONCLUSION: We conclude that the bystander effect in murine neuroblastoma depends on immune response and is enhanced by neuro-2a/IL-2.
Animals ; Antibodies, Monoclonal ; Bystander Effect* ; Connexin 43 ; Gap Junctions ; Genetic Therapy* ; Immunohistochemistry ; Mice ; Neuroblastoma* ; Simplexvirus ; 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

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*

Adipose-derived stem cells (ASCs) are believed to have potential use for treating many illnesses. Most cells, including ASCs, are generally cultured in medium containing fetal bovine serum (FBS). However, FBS, which could induce an immune response or infection, is not recommended for clinical applications. In the present study, we evaluated the morphology, proliferation rate, and characterization of rabbit ASCs grown in medium containing autologous serum (AS) and compared these cells to ones cultured with FBS. Morphological changes were monitored by microscopy and flow cytometry. Proliferation rates were assessed with cell counting and ASC phenotypes were characterized by flow cytometry using representative surface markers (CD44 and CD45). Expression of epidermal growth factor, brain-derived neurotrophic factor, and vascular endothelial growth factor was measured by reverse transcription-polymerase chain reaction. Results of our study showed that ASCs had a greater expansion rate in AS without developing morphological heterogeneity than cells grown in FBS. AS-cultured ASCs expressed representative growth factors, CD44 but not CD45, similar to cells cultured in FBS. Expression levels of some growth factors were different between AS and FBS. In conclusion, our findings indicated that AS could potentially be used as a culture medium supplement for the expansion of autologous ASCs.
Brain-Derived Neurotrophic Factor ; Bystander Effect ; Cell Count ; Epidermal Growth Factor ; Flow Cytometry ; Intercellular Signaling Peptides and Proteins ; Microscopy ; Phenotype ; Population Characteristics ; Stem Cells ; Vascular Endothelial Growth Factor A

BACKGROUND: Recombinant adenovirus hold promise as vectors to carry therapeutic genes for several reasons: 1) they can infect both dividing and non-dividing cells ; 2) they have the ability to directly transduce tissues in vivo; 3) they can easily be produced in high titer ; and 4) they have an established record of safety as vaccination material. However, one of the major limitation in the use of adenoviruses is that transgene expression is quite short because adenovirusees insert their DNA genome episomally rather than by chromosomal integration, and an immune response against the virus destroys cells expressing the therapeutic gene. Since sodium butyrate has been reported to induce adenovirus-mediated gene expression, we hypothesized that treatment of tumor cells, transduced with herpes simples virus thymidine kinase(HSVtk) gene using adenoviral vector, with butyrate could augment the effect of gene therapy. METHODS: We transduced HSVtk gene, driven by the cytomegalovirus promoter, into REN cell line(human mesothelioma cell line). Before proceeding with the comparison of HSVtk/ganciclovir mediated bystander killing, we evaluated the effect of butyrate on the growth of tumor cells in order to rule out a potential antitumor effect of butyrate alone, and also on expression of HSVtk gene by Western blot analysis. Then we determined the effects of butyrate on bystander-mediated cell killing in vitro. RESULTS: There was no inhibition of growth of cells exposed to butyrate for 24 hours at a concentration of 1.5mM/L. Toxic effects were seen when the concentration of butyrate was greater than 2.0mM/h Gene expression was more stable and bystander effect was augmented by butyrate treatment of a concentration of 1.5mM CONCLUSION: These results provide evidence that butyrate can augment the efficiency of cell killing with HSVtk/GCV system by inducing transgene expression and may thus by a promising new approach to improve responses in gene therapy using adenoviral vectors.
Adenoviridae ; Blotting, Western ; Butyrates* ; Butyric Acid ; Bystander Effect ; Cytomegalovirus ; DNA ; Gene Expression ; Genetic Therapy* ; Genome ; Herpes Simplex* ; Homicide ; Mesothelioma ; Phosphotransferases* ; Simplexvirus* ; Thymidine ; Transgenes ; Vaccination

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