[Effect of adenovirus-mediated TK/GCV gene therapy in combination with TNF-alpha against murine bladder cancer cells in vitro].
OBJECTIVE
To investigate the cell-killing effect of adenovirus-mediated TK-ganciclovir (GCV) gene therapy in combination with tumor necrosis factor-alpha (TNF-alpha) against murine bladder carcinoma cells in vitro.


METHODS
Murine bladder carcinoma MB49 cells were transfected with the adenoviral vector containing TK gene and green fluorescent protein (GFP) gene. The transfection efficiency was observed and the TK gene expression in the transfected cells was detected by RT-PCR. The survival rate of MB49 cells in response to TNF-alpha treatment and that of the TK gene-transfected cells after treatment with GCV and GCV+TNF-alpha were determined by MTT assay. The apoptosis of the cells after the treatments was analyzed by flow cytometry.


RESULTS
In cells transfected with TK gene, the cell inhibition rate increased gradually with the increment of GCV and TNF-alpha concentration. GCV in combination with TNF-alpha resulted in significantly increased killing efficiency of the cells as compared with GCV or TNF-alpha treatment alone, and the effect of the combined treatment was enhanced as the TNF-alpha concentration increased. GCV treatment (50 microg/ml) alone produced a cell killing rate of (24.39-/+1.10)%, and when combined with 5 microg/ml TNF-alpha, the rate was increased to (40.05-/+0.97) %, and further to (65.47-/+0.67) % when TNF-alpha concentration increased to 20 microg/ml. Flow cytometry revealed obvious apoptosis of the cells 8 h after treatments with TK/GCV, TNF-alpha, or TK/GCV+TNF-alpha, and the combined treatment resulted in the highest cell apoptotic rate.


CONCLUSIONS
TK/GCV in combination with TNF-alpha can enhance the effect of suicide gene therapy against murine bladder carcinoma cells and effectively induce apoptosis of the cells.
Shi
Xiang-hua
XH
Department of Urologic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China sxh96@ hotmail.com
Tan
Wan-long
WL
Zhu
Wen-hui
WH
Liang
Zhong-kun
ZK
DU
Yue-jun
YJ
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D015774:Ganciclovir / Q000378:metabolism / Q000494:pharmacology*; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology
D000998:Antiviral Agents; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase; D015774:Ganciclovir