Kinase domain insert containing receptor promotor controlled suicide gene system kills human umbilical vein endothelial cells.
OBJECTIVE
To evaluate the killing effect of double suicide gene mediated by adenovirus and regulated under kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells.


METHODS
By PCR technology, human KDR promoter gene, Escherichia coli (E. coli) cytosine deaminase (CD) gene and the herpes simple virus-thymidine kinase (TK) gene were cloned. Plasmid pKDR-CDglyTK was constructed with them. Then, a recombinant adenoviral plasmid pAdKDR-CDglyTK was constructed in a "two-step transformation protocol". The newly constructed plasmids were transfected to 293 packaging cells to grow adenoviruses, which were further propagated and purified. Human umbilical vein endothelial cells (HUVEC) were infected with a different multiplicity of infection (MOI) of resultant recombinant adenovirus, the infection rate was measured with the aid of (GFP) expression. Infected cells were cultured in culture media containing different concentrations of (GCV) and/or 5-(FC), and the killing effects were measured.


RESULTS
Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed, and they infected HUVEC cells efficiently. Our data indicated that the infection rate was relevant to MOI of recombinant adenoviruses. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of recombinant adenoviruses. Our data also indicated that the two prodrugs used in combination were much more effective on killing transgeneic cells than GCV or 5-FC used alone.


CONCLUSIONS
Prodrug/KDR-CDglyTK system is effective on killing HUVEC cells, its killing effect correlates to the concentration of prodrugs and recombinant adenovirus' MOI. Combined use of the two prodrugs confers better killing effects on transgeneic cells.
Huang
Zong-Hai
ZH
Department of General Surgery, Zhujiang Hospital, First Military Medical University, Guangzhou 510282, Guangdong Province, China. yangwenyugang@vip.sina.com
Yang
Wen-Yu
WY
Cheng
Qi
Q
Yu
Jing-Long
JL
Li
Zhou
Z
Tong
Zong-Yan
ZY
Song
Hui-Juan
HJ
Che
Xiao-Yan
XY
D000256:Adenoviridae / Q000235:genetics; D016923:Cell Death / Q000235:genetics*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000502:physiology*; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D014471:Umbilical Veins / Q000166:cytology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics*
D040301:Vascular Endothelial Growth Factor Receptor-2