We have previously reported that direct transfer of the TNF-related apoptosis-inducing ligand (TRAIL) gene resulted in an apoptotic bystander effect, and that this bystander effect was not transferable with cell culture media. To further characterize its mechanism we tested the bystander effect of TRAIL in the human ovarian cancer cell line DOV13, human lung cancer cell line A549, human hepatoma cell line Hepa G2, human breast cancer cell line MDA-MB231 and human colon cancer cell lines Lovo and DLD1. The bystander target cells were transduced with an adenovector expressing the lacZ gene (Ad/CMV-LacZ), while the effector cells were transduced with an adenovector expressing the green fluorescent protein (GFP)/TRAIL fusion gene. Effector and target cells were then cocultured in the same well with or without effector and target cell contact. In all the cell lines tested, target cells were killed if effector and target cell contact was permitted. However, no bystander effect occurred if effector and target cell contact was prevented. Furthermore, the bystander effect and apoptosis induction of TRAIL was dramatically reduced if cells were seeded at a very low density. Moreover, in all the cell lines tested, no detectable soluble TRAIL was found in media from the TRAIL-expressing cell cultures. Together, our results demonstrated that release of soluble TRAIL from transfer of the wild-type TRAIL gene is minimal, and that the bystander effect of the TRAIL gene is mainly mediated by membrane-bound TRAIL on the surface of transduced cells.

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