title: Double suicide genes selectively kill human umbilical vein endothelial cells.
abstract: BACKGROUND
  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.
  
  
  METHODS
  Human 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.
  
  
  RESULTS
  Recombinant 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.
  
  
  CONCLUSIONS
  AdKDR-CDglyTK/double prodrog system may be a useful method for suppressing tumor angiogenesis.
authors.affiliation: Department of Thoracic Surgery, West China Hospital, Sichuan University, Chengdu 610041, PR China.
authors.forename: Weiguo
authors.initials: W
authors.lastname: Jia
authors.forename: Longyong
authors.initials: L
authors.lastname: Mei
authors.forename: Yanping
authors.initials: Y
authors.lastname: Wang
authors.forename: Lunxu
authors.initials: L
authors.lastname: Liu
authors.forename: Guowei
authors.initials: G
authors.lastname: Che
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects* / Q000821:virology; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009369:Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D009389:Neovascularization, Pathologic; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D013937:Thymidine Kinase; D040301:Vascular Endothelial Growth Factor Receptor-2