Detecting Integrin αv To Evaluate The Susceptibility Of Tumor Cells To Adenovirus Vector

Recombinant human adenoviral(Ad) vectors are attractive gene delivery vehicles for cancer gene therapy, because of the following reasons: Ad vectors are easy to generate and manipulate. They are relatively stable and can be readily obtained in high titers. They do not integrate into the host cell genome. Therefore, the risk of provoking mutations in host genes is extremely low. They can infect a broad range of cell types, including both dividing and non-dividing cells. They have 36kb that is suitable for the development of large-capacity vectors with minimal viral sequence. Adnoviral vector-mediated gene transfer was highly effective in animal models,but large differences regarding transduction efficiencies among different cell lines and between in vitro and in vivo gene transfer have been reported. The results of adenovirus-mediated cancer gene therapy have been disappointing, with only limited efficacy being observed in preclinical and clinical studies. This is especially crucial as efficient transduction of target cells is one of the prerequisites for successful transgene expression and therapeutic efficiency for patients. Therefore, evaluating susceptibility of tumor cells to adenoviral vectors prior to adenovirus-based gene therapy may be important. The mechanism of adenoviral attachment and internalization indicated that the cells should expressed sufficient coxsackievirus and adenovirus receptor (CAR) and integrin αv to obtain high transdution efficiency. The aim of this study is analyzing the relation between integrin αv expression and transduction efficiency of adenoviral vectors. Methods: Eight human tumor cell lines were tested. First, the expression levels of integrin αvβ3 on each cell lines were examined by immunohistochemical staining. Integrin αv mRNA were detected by in situ hybridization. Adenoviral transduction efficiency was determined with an E1-deleted adenovirus type 5 expressing beta-galactosidase under a CMV promoter AdCMVLacZ. The cells(5×104) were seeded into a 24-well plate. On the following day, they were transduced with AdCMVLacZ at 50 MOI for 3h. After 48h in culture, LacZ expression in the cells were measured using X-gal staining method. Finally, the transduction efficiency was compared to the results of immunihistochemical staining and in situ hybridization. Results: 1. Overall, adenovirus transduction efficiency increased with increasing titers of AdCMVLacZ. At a given titer of 50 MOI, transduction efficiency varied from 90.2% to 7.6% among the individual cell lines. 2. The transduction efficiency could be reduced by using GRGDSP before infecting cells with AdCMVLacZ. 3. After histochemical staining to the cells, significant amounts of αvβ3 were detected on A549 and SMMC-7721 cells. In contrast, no αvβ3 were detected on Hela and M21-L cells. The trasduction efficiency increased in the cells with high αvβ3 expression. 4. Integrin αv mRNA were detected by in situ hybridization. Integrin αv mRNA were...