Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells.
OBJECTIVE
To study the selective killing of human umbilical vein endothelial cells (HUVECs) by a double suicide gene under the regulation of a kinase domain insert containing receptor (KDR) promoter and mediated by an adenoviral gene vector.


METHODS
Human KDR promoter was cloned by polymerase chain reaction (PCR), and two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified. HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses (AdKDR-CDglyTK and AdCMV-CDglyTK) respectively, and the infection rates were estimated by detection of green fluorescent protein (GFP) expression. Infected cells were cultured in culture media containing different concentrations of 5-fluorocytosine (5-FC) and ganciclovir (GCV), and the killing effects were measured.


RESULTS
The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs: HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMV-CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK (P < 0.001).


CONCLUSIONS
Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.
Yang
Wen-Yu
WY
Department of General Surgery, Zhujiang Hospital, First Military Medical University, Guangzhou 510282, Guangdong Province, China. yangwenyugang@vip.sina.com
Huang
Zong-Hai
ZH
Lin
Li-Jun
LJ
Li
Zhou
Z
Yu
Jing-Long
JL
Song
Hui-Juan
HJ
Qian
Yong
Y
Che
Xiao-Yan
XY
D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology*; D005821:Genetic Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D011355:Prodrugs; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology*
D011355:Prodrugs