[Specific antitumor effect of adeno-associated virus vector carrying TRAIL gene under the control of hTERT promoter].
OBJECTIVE
Adeno-associated virus (AAV) has been widely used in tumor gene therapy. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a safe and potent anti-tumor gene which could induce apoptosis of many tumor cells. This study was to use tumor-specific promoter hTERT to construct AAV-hTERT-TRAIL, and explore its antitumor effect and mechanism in vitro.


METHODS
Purified AAV-hTERT-TRAIL was obtained after co-transfection of HEK293 with pAAV-hTERT-TRAIL and two other help plasmids. After transfection of AAV-hTERT-TRAIL into three tumor cell lines, SW620, HepG2, A549 and two normal cell lines, NHLF and MRC5, the expression of TRAIL was detected by RT-PCR, Western blot and immunohistochemistry (IHC); the influence of AAV-hTERT-TRAIL transfection on cell proliferation was evaluated using MTT assay. Activation of caspase-3 and PARP was measured by Western blot. Cell apoptosis was assessed using ELISA and flow cytometry.


RESULTS
AAV-hTERT-TRAIL was successfully packaged in HEK293 cells. After AAV-hTERT-TRAIL infection, specific expression of TRAIL was detected in three tumor cell lines, but not in two normal cell lines. Cell proliferation rates in SW620, A549, HepG2, NHLF and MRC5 cells were 41.55%, 44.29%, 49.95%, 84.59% and 87.22%, respectively after transfection of AAV-hTERT-TRAIL at a multiplicity of infection (MOI) of 100 for 96 h. AAV-hTERT-TRAIL activated caspase-3 apoptotic pathway and induced apoptosis in tumor cell lines, but not in normal cell lines.


CONCLUSIONS
hTERT increases selectivity and safety of AAV vector. hTERT promoter controls the expression of anti-tumor genes to specifically induce death of tumor cells.
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China.
Rong
R
Qi
Ying
Y
Cai
Bing-Hua
BH
Li
Zhi-Xin
ZX
Lin
Jin-Fa
JF
Gu
D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D000229:Dependovirus / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism*; D019098:Telomerase / Q000235:genetics*; D014162:Transfection
D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C509186:TERT protein, human; D019098:Telomerase; C505407:CASP3 protein, human; D053148:Caspase 3