The bystander effect exerted by tumor cells expressing the herpes simplex virus thymidine kinase (HSVtk) gene is dependent on connexin expression and cell communication via gap junctions

To elucidate the role gap junctions play in the bystander effect, we examined the cytotoxic effect of herpes simplex virus thymidine kinase (HSVtk) modified tumor cells on gap junction communication-deficient tumor cells and their connexin transfectants. Communication competent Walker 256 tumor cells engineered to express the HSVtk gene (Walker-tk⁺) when cocultured with N2A mouse neuroblastoma and PC12 rat pheochromocytoma cells with absent endogenous junctional conductance showed no bystander cytotoxicity. Transfection of N2A cells with the rat connexin37 gene (5Q) and PC12 cells with the human connexin43 gene rendered them susceptible to bystander cell death. Additionally, communication-deficient N2A cells transfected with the HSVtk gene failed to exert a bystander effect, whereas N2A transfectants coexpressing the connexin37 and HSVtk genes (5Qtk⁺ cells) exerted bystander cytotoxicity on gap unction communication-competent 5Q but not on communication-deficient N2A cells in vitro. In vivo experiment also showed tumor growth inhibition of communication-competent 5Q but not communication-incompetent N2A cells by 5Qtk cells. In conclusion, these results indicate that in several cellular environments the bystander effects is dependent on connexin expression and gap junctional communication between HSVtk-positive and HSVtk-negative cells.