科研之友
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摘要
Background Recombinant adenoviruses (Ad) have been employed as vectors for a wide variety of gene therapy applications, but their use has been hindered by problems relating to efficacy and safety. The efficiency of Ad-mediated gene transfer depends on the interaction of the fibre and penton base proteins with their corresponding cell receptors. Ad infection is initiated by the formation of a high affinity complex between the fibre protein and a host cell protein that for most Ad serotypes is CAR (the coxsackie B virus and Ad receptor). A second molecule, the MHC class I, may also be involved in Ad type 2 and Ad type 5 uptake. Ad internalization results from the interaction of the penton base protein with cell surface integrins alpha (v)beta (3) and alpha (v)beta (5). In this study, we addressed the interaction between Ad type 5 (Ad5) and its receptors on lung derived adenocarcinoma cells in culture.
Methods Using flow cytometry, we determined the level of expression of attachment and internalization receptors that are expressed on the cell surface of A549, H322 and H441 lung-derived adenocarcinoma cells in culture. The level of alpha (v)beta (1) cell surface integrin was assessed by immunoprecipitation. Measuring the level of luciferase gene expression at different viral titres quantitated Ads uptake by these cells. The kinetics of binding of Ads fibre knobs to A549, H322 and H441 cells was assessed in direct binding studies using I-125 labelling of purified recombinant Ads fibre-knob domains. In order to assess the functionality of integrins, adhesion assays were performed in the presence or absence of activators of integrin function. In competition experiments, prior to exposure to the virus, the cells were preincubated with purified recombinant Ads fibre-knob domains, function blocking anti-integrin antibodies, or integrin activating agents, prior to the introduction of luciferase expressing Ads.
Results We found that Ads-mediated gene transfer in A549, H322 and H441 adenocarcinoma cells in culture is highly variable and that this variation correlates with specific binding of Ads fibre-knob domain binding to the cell surface. We also found, for the first time, that Ads infection is mediated by integrin alpha (v)beta (1) and that functional activation of beta (1) integrin by means of the specific anti-beta (1) monoclonal antibody, TS2/16, induced increased A549 cell adhesion to fibronectin and vitronectin and also enhanced Ads uptake by these cells.
Conclusions Our studies demonstrate that the Ads fibre-knob domain interaction with CAR represents a major determinant of Ads-mediated gene transfer to lung-derived adenocarcinoma cells in culture. The finding that integrin alpha (v)beta (1) is involved in Ads infection has implications for the use of recombinant Ads vectors for cancer gene therapy, since alpha (v)beta (1) is expressed at high levels and acts as an alternative vitronectin receptor in many epithelial and some melanoma tumours which express no alpha (v)beta (3) and constant low levels of alpha (v)beta (5). The fact that the beta (1) integrin-activating antibody TS2/16 can enhance alpha (v)beta (1)-mediated Ads infection suggests that the efficacy of Ad5-mediated gene transfer might be influenced not only by the level of cell surface expression of integrins but also by their state of activation.
- 出版日期2001-12
