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

PURPOSE: We have previously cloned three enhancer factor genes encoding proteins that bind to long terminal repeats (LTRs) of Rous sarcoma virus. Among these genes, RSV- EF-I gene is expressed in rat hepatoma tissues and several proliferating cell lines but not in normal rat liver tissues. We have isolated the human homologue of RSV-EF-I gene and examined its expression in human hepatocellular carcinoma tissues. MATERIALS AND METHODS: We have screened the human genomic library and cDNA library of Hep G2 cell line derived from human hepatocellular carcinoma to isolate the human homologue of RSV-EF-I gene. RESULTS: We have isolated one cDNA clone containing about 1.5 kb insert and sequenced. Sequence analysis reveals that this human homologue of RSV-EF-I gene has a high similarities to human YB-1 mRNA, human DNA-binding protein B (dbpB) gene and other Y-box protein genes. It is expressed in human hepatocellular carcinoma but very slightly in normal human liver tissues in Northern blot analysis. CONCLUSION: Our data suggest that the human homologue of RSV-EF-I gene presumably belongs to Y-box protein family genes and plays a role in the transformation of the human hepatoma cells.
Animals ; Blotting, Northern ; Carcinoma, Hepatocellular* ; Cell Line ; Clone Cells ; DNA, Complementary ; Gene Library ; Genomic Library ; Hep G2 Cells ; Humans* ; Liver ; Rats ; RNA, Messenger ; Rous sarcoma virus* ; Sarcoma, Avian* ; Sequence Analysis ; Terminal Repeat Sequences

PURPOSE: The poor prognosis of advanced bladder cancer requires the investigation of novel treatment modalities. In this study, we investigated the suicide gene therapy for bladder cancer, using the adenovirus-mediated expression of Escherichia coli cytosine deaminase (CD) in conjunction with the prodrug 5-fluorocytosine (5-FC). MATERIALS AND METHODS: A replication-deficient recombinant adenovirus, which contains the Rous sarcoma virus (RSV) promoter driving the expression of CD, (Ad-RSV-CD) was constructed. In vitro cell-killing assay, using Ad-RSV-CD (20 MOI) plus 5-FC (500muM), was performed in bladder cancer cell lines, HT-1376, UM-UC-3 and NBT-II. The CD enzymatic activity was measured in the Ad-RSV-CD (20 MOI) infected cells, and the concentrations of 5-fluorouracil (5-FU) yielding an IC50 were calculated for those cells. RESULTS: 5-FU dose response curve showed that IC50 of NBT-II was 0.8muM, HT-1376 1.0muM and UM-UC-3 5.1muM at day 6. The CD enzymatic activities of the Ad-RSV-CD infected UM-UC-3, HT-1376 and NBT-II cells were 5696, 4655, 1766 pmole/1x10(6) cells, respectively. Whereas the administration of 5-FC (500muM) or Ad-RSV-CD (20 MOI) alone demonstrated no cytotoxicity to cells, Ad-RSV-CD/5-FC exhibited a significant cytotoxic effect in the cells, especially the UM-UC-3 and HT-1376. CONCLUSIONS: Ad-RSV-CD/5-FC suicide gene therapy is effective for bladder cancer cells in cell cultures, suggesting this approach may have potential as a strategy for the treatment of bladder cancer.
Adenoviridae* ; Cell Culture Techniques ; Cell Line ; Cytosine Deaminase* ; Cytosine* ; Escherichia coli* ; Escherichia* ; Flucytosine ; Fluorouracil ; Genetic Therapy* ; Inhibitory Concentration 50 ; Prognosis ; Rous sarcoma virus ; Suicide* ; Urinary Bladder Neoplasms* ; Urinary Bladder*