[Killing effect of Ad.TERT-TRAIL on tumor cell lines and its mechanism].
OBJECTIVE
Oncolytic adenovirus Ad.TERT, a novel tumor-specific proliferating virus, has been constructed by replacing normal promoter of mild-type adenovirus E1A with promoter of human telomerase reverse transcriptase (hTERT). In vitro and in vivo experiments confirmed that Ad.TERT has antitumor effect. This study was to construct Ad.TERT-TRAIL through inserting apoptosis gene trail into Ad.TERT, and explore its antitumor effect and mechanism.


METHODS
Plasmid pZhTERT-trail and adenovirus packaging plasmid pBHGE3 were homologously recombined in HEK293 cells to construct Ad.TERT-TRAIL. Ad.TERT-TRAIL was identified by polymerase chain reaction (PCR), and confirmed by Western blot. Killing effects of Ad.TERT-TRAIL and Ad.TERT on 3 tumor cell lines, SW620, BEL-7404 and Bcap-37, and a normal cell line NHLF were detected by crystal violet dye method or MTT assay. Expression of Caspase-3 in Ad.TERT-TRAIL-, and Ad.TERT-transfected SW620 cells was detected by Western blotu cell apoptosis was detected by flow cytometry (FCM).


RESULTS
The 860 bp-length trail gene has been amplified by PCR. Western blot showed trail and E1A only expressed in tumor cells, which confirmed the successful construction of Ad.TERT-TRAIL. Killing effects of Ad.TERT-TRAIL on tumor cells were 10-100 times as strong as that of Ad.TERTu while both of them had little effects on normal cells. After 3 days infection (100 multiple of infection, MOI), survival rate of Ad.TERT-TRAIL-infected SW620 cells was 4%, but that of Ad.TERT-infected SW620 cells was 56%u both viruses had little effects on NHLF cells. Expression of Caspase-3 was higher in Ad.TERT-TRAIL-infected SW620 cells than in Ad.TERT-infected SW620 cells. Apoptosis rate of Ad.TERT-TRAIL-infected SW620 cells was 4 times as high as that of Ad.TERT-infected SW620 cells.


CONCLUSIONS
Ad.TERT-TRAIL has much stronger antitumor effect than Ad.TERT. Its effect might relate with inducement effects of trail gene on expression of Caspase-3, and apoptosis of tumor cells.
Tang
Yun-Xia
YX
Xin Yuan Institute of Medincine and Biotechnology, School of Life Science, Zhejiang Sci-Tech University, Hangzhou, Zhejiang, 310018, P.R.China. xyliu@sibs.ac.cn.
Chen
Yu
Y
Gu
Jin-Fa
JF
Qi
Rong
R
Zou
Wei-Guo
WG
Jiang
Lin-Lan
LL
Liu
Xin-Yuan
XY
D000256:Adenoviridae / Q000235:genetics*; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D004622:Embryo, Mammalian; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D053221:TNF-Related Apoptosis-Inducing Ligand; D019098:Telomerase / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics*
D051017:Apoptosis Regulatory Proteins; D004268:DNA-Binding Proteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; C509186:TERT protein, human; D019098:Telomerase; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases