Abstract
BackgroundTo construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells.MethodsHuman KDR promoter, Escherichia coli (E. coli) cytosine deaminase (CD) gene and the herpes simplex virus-thymidine kinase (TK) gene were cloned using polymerase chain reaction (PCR). Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304) and KDR-negative liver cancer cell line (HepG2) were infected with the recombinant adenoviruses at different multiplicity of infection (MOI). The infection rate was measured by green fluorescent protein (GFP) expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV) and/or 5-fluorocytosine (5-FC). The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL) staining.ResultsRecombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs.ConclusionAdKDR-CDglyTK/double prodrog system may be a useful method for suppressing tumor angiogenesis.
Highlights
To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells
We proposed that the use of KDR promoter in combination with double suicide genes that were activated in the tumor’s vascular endothelial cells would provide us with high levels of tumor specificity
KDR promoter region was amplified from the genomic DNA of ECV304 cells by polymerase chain reaction (PCR)
Summary
To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells. Anti-angiogenic therapy has been proved to be a rational approach in the treatment of solid tumors [1]. The advantages of anti-angiogenic approach include new vessels targeting, the lack of mutations in the endothelial cells, and the amplification effect on tumor killing [2]. Targeting endothelial cells by gene therapy was hampered by the inefficiency of the vascularspecific promoters. We used a modified kinase domain insert containing receptor (KDR) promoter to direct gene expression in endothelial cells. KDR gene is strictly expressed only in vascular endothelial cells.
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