title: Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells.
abstract: OBJECTIVE
  To investigate the killing efficiency of a recombinant plasmid containing a thymidine kinase (TK) domain insert driven by the vascular endothelial growth factor receptor 2 (VEGFR2) promoter (KDR) on vascular endothelial cells.
  
  
  METHODS
  The KDR-TK fragment was extracted from pBluescript II KDR-TK plasmid by enzymatic digestion with XhoI and SalI. The enhanced green fluorescence protein (EGFP) carrier was extracted from pEGFP by the same procedure. The KDR-TK was inserted into the pEGFP carrier to construct pEGFP-KDR-TK. Using ultrasound irradiation and microbubble, pEGFP-KDR-TK was transferred into human umbilical vein endothelial cells (HUVECs). The transient infection rate was estimated by green fluorescent protein (GFP) expression. Transfected HUVECs, non-transfected HUVECs, and HepG2 cells were cultured in the presence of different concentrations of ganciclovir (GCV), and the killing efficacy of HSV-TK/GCV was analyzed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay.
  
  
  RESULTS
  The recombinant pEGFP-KDR-TK was successfully constructed by inserting the KDR-TK fragment into the pEGFP carrier. Transfected HUVECs showed cytoplasmic green fluorescence, and the transient transfection rate was about 20.3%. Pools of G418-resistant cells exhibited a higher sensitivity to the prodrug/GCV compared to non-transfected HUVECs or non-transfected HepG2 cells, respectively.
  
  
  CONCLUSIONS
  KDR promoter and the suicide gene/prodrug system mediated by diagnostic ultrasound combined with microbubble can significantly kill HUVECs. Such therapy may present a novel and attractive approach to target gene therapy on tumor vessels.
authors.affiliation: Department of Medical Ultrasonics, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, Guangdong Province, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Hui-Xiong
authors.initials: HX
authors.lastname: Xu
authors.forename: Ming-De
authors.initials: MD
authors.lastname: Lu
authors.forename: Qing
authors.initials: Q
authors.lastname: Tang
mesh_terms: D006528:Carcinoma, Hepatocellular; D016923:Cell Death / Q000502:physiology; D045744:Cell Line, Tumor; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D008113:Liver Neoplasms; D011401:Promoter Regions, Genetic / Q000502:physiology; D013937:Thymidine Kinase / Q000235:genetics*; D014471:Umbilical Veins / Q000166:cytology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics*
chemical_list: D013937:Thymidine Kinase; D040301:Vascular Endothelial Growth Factor Receptor-2