[Suppression of osteosarcoma in vitro by coexpression of antisense VEGF165 cDNA and thymidine kinase gene].
OBJECTIVE
To investigate the effect of VEGF expression in osteosarcoma cell line and the target killing effect of HSV1-TK/GCV system on transfected osteosarcoma cells under hypoxia conditions.


METHODS
Eukaryotic expression plasmid with HRE promoter was constructed to express the antisense VEGF165 cDNA and Hygromycin phospho-transferase-thymidine kinase (HyTK) fusion gene. The recombinant vectors were then transfected into osteosarcoma cell line MG63 with lipofectin mediated gene transfer methods. PCR and RT-PCR were used to confirm the presence and expression of TK gene. The sensitivity of transfected cells to GCV and "bystander effect (BSE)" of HSV1-TK/GCV system under normoxia or hypoxia conditions were measured by MTT assay and mixed co-culture experiment. The expression of VEGF protein was detected by ELISA under hypoxia condition. Cell cycle phase distribution was determined by flow cytometry. In addition, electromicroscopy was used to document ultrastructural alterations.


RESULTS
The eukaryotic expression vector pBI-HRE-AsVEGF165 -HyTK was constructed successfully. The transfected cell line MG63TV was established and confirmed by PCR and RT-PCR of the presence of transgene and its mRNA expression. GCV was toxic to transfected cells in a concentration-dependent manner. The sensitivity to GCV toxicity was 100 times higher under hypoxia condition than that under normoxic condition. The mixed culture experiments showed that the "bystander effect" was enhanced significantly under hypoxia condition. VEGF expression of transgene cells under hypoxia condition decreased 50% compared to that of normal condition. Under hypoxia and GCV, DNA synthesis of MG63TV cells was inhibited along with an increase of cells at G0 approximately G1 phase, apoptosis and necrosis.


CONCLUSIONS
Antisense VEGF expression driven by HRE promoter in combination with hypoxia can provide a target inhibition of VEGF expression in human osteosarcoma cells, with an enhanced selective killing effect and BSE of the HSV-TK/GCV system. The double-gene co-expression system in study provides experimental basis for therapy against osteosarcoma by a synchronous antiangiogenic and suicide gene approach.
Wang
Yan
Y
Department of Pathology, Sun Yat-sen Medical College, Sun Yat-sen University, Guangzhou 510089, China. wyan_sky@163.com
Qiao
Hui
H
Peng
Ting-Sheng
TS
Li
Yang
Y
Zhang
Meng
M
Liang
Hui-Zhen
HZ
Qiu
Ju-Shi
JS
D017209:Apoptosis; D001859:Bone Neoplasms / Q000378:metabolism / Q000473:pathology; D024201:Bystander Effect; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004273:DNA, Neoplasm / Q000096:biosynthesis; D015774:Ganciclovir / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics*; D020319:Oligodeoxyribonucleotides, Antisense; D012516:Osteosarcoma / Q000378:metabolism / Q000473:pathology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013937:Thymidine Kinase / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D004273:DNA, Neoplasm; D051793:Hypoxia-Inducible Factor 1; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D013937:Thymidine Kinase; D015774:Ganciclovir