[Construction and identification of recombinant vectors carrying herpes simplex virus thymidine kinase genes expressed in vascular endothelial cells].
OBJECTIVE
Blocking tumor angiogenesis is a promising tumoricidal approach. Herpes simplex virus-thymidine kinase (HSV-tk) suicide gene could effectively damage neoplastic vascular endothelial cells. However, the killing effect of currently used HSV-tk system is non-specific due to employing cytomegalovirus (CMV) as its promoter. Kinase domain insert containing receptor (KDR), a kind of vascular endothelial growth factor receptor, is highly expressed on tumor vascular endothelial cells, but lowly expressed on normal tissues. This study was to construct a recombinant adenovirus containing KDR promoter-mediated HSV-tk suicide gene, and analyze its specific expression on vascular endothelial cells.


METHODS
KDR-tk fragment was subcloned into corresponding restriction sites of vector with directional cloning method using pAdeasy system to construct recombinant adenoviral plasmid containing KDR promoter-controlled HSV-tk gene (AdKDR-tk). After packaged and amplified in 293 cells, the virus was used to infect KDR-expressed human umbilical venous endothelial cells (HUVECs) and KDR-unexpressed liver cancer cell line HepG2. After administration of ganciclovir (GCV), proliferation of gene-transfected HUVECs and HepG2 cells was evaluated by MTT assay.


RESULTS
According to the results of restriction digestion, reverse transcription-polymerase chain reaction (RT-PCR) and PCR, the length, position and orientation of inserted genes were correct. The titer of the recombinant adenovirus was 1 x 10 (10) pfu/ml. Under infection index of 100, with the concentration of GCV increased from 0 to 50 microg/ml, the survival rates of AdKDR-tk-transfected HUVECs and HepG2 cells decreased from 100% to (28.94+/-5.67)% and (75.45+/-2.91)%, respectively (P<0.01).


CONCLUSIONS
Adenovirus-mediated transfection of KDR promoter-HSV-tk gene could direct endothelial specific gene expression.
Department of Hepatobilliary and Pancreatic Surgery, Shenzhen Hospital, Peking University, Shenzhen, Guangdong 518036, P. R. China. ninliu@163.com
Xiao-Ping
XP
Liu
Bao-Jin
BJ
Li
Chao
C
Zhang
D000256:Adenoviridae / Q000235:genetics; D000998:Antiviral Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D015774:Ganciclovir / Q000494:pharmacology*; D005822:Genetic Vectors; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology*; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014471:Umbilical Veins / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*
D000998:Antiviral Agents; D011994:Recombinant Proteins; D013937:Thymidine Kinase; D040301:Vascular Endothelial Growth Factor Receptor-2; D015774:Ganciclovir