[Enhanced transduction efficiency of adeno-associated virus on cancer cells with the help of low dose adenovirus].
OBJECTIVE
To investigate whether adeno-associated virus (AAV) could enhance its infection efficiency on cancer cells when combined with non-replicable adenovirus (Ad-null) in vitro as well as in vivo and to study its underlying mechanisms.


METHODS
AAV2 particle was added into NCI-H460 tumor cell lines alone or in combination with different amount of adenovirus. 1 to 7 days after transduction, cells were observed and recorded with fluorescence microscope, the expression levels of report gene EGFP in tumor cells were examined by using flow cytometry and Western blotting, the expression of report genes luciferase was analyzed with luminometer to obtain the relative light units. After the establishment of tumor model, the nude mouse were administrated with AAV2 or AAV2 + Ad-null in tumors, and then tested their infection efficiency and expression levels with roperscientific bioluminescence tumor imaging system.


RESULTS
The results obtained with the help of flow cytometry and luciferase assay suggested that the infection efficiency of AAV2 was enhanced significantly when combined with low dose Ad-null in vitro, the infection efficiency of AAV2 alone was 6.4% and it reached 55.2% when combined with 10 MOI Ad-null, Western blotting assay demonstrated that the protein expression level of reporter gene in tumor cells enhanced when combined with 10 MOI Ad-null compared with AAV2 infection alone, and the enhancement of reporter gene expression was observed in a concentration-dependent manner; real-time PCR analysis confirmed that Ad-null enhanced the mRNA level of AAV2-EGFP but not the copies of genomic DNA of AAV2-EGFP. Ad-null significantly augmented the infection efficiency when tested on NCI-H460 tumor model. With the help of Ad-null, the signal of luciferin in nude mouse was 4.5 times more than that of control.


CONCLUSIONS
The infection efficiency of AAV was enhanced significantly when combined with low dose Ad-null in vitro and in vivo, and it offers basis for further study of gene therapy by AAV.
Li
Hui-ming
HM
Central Experimental Laboratory, the First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China.
Wang
Feng
F
Wei
Fang
F
Dong
Xiao-yan
XY
Wang
Hui-ping
HP
Qiu
Wei
W
Zhang
Ju-feng
JF
Chen
Xia-fang
XF
Wu
Xiao-bing
XB
Huang
Qian
Q
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D000229:Dependovirus / Q000235:genetics*; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D014183:Transplantation, Heterologous
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases