IN-VITRO EVIDENCE THAT METABOLIC COOPERATION IS RESPONSIBLE FOR THE BYSTANDER EFFECT OBSERVED WITH HSV TK RETROVIRAL GENE-THERAPY

Tumor cells transduced with a retroviral vector expressing a herpes vi rus thymidine kinase (HSV tk) gene are rendered sensitive to the antih erpetic drug, ganciclovir. The bystander effect refers to the observat ion that not all cells need be transduced to eradicate the cell popula tion by treatment with ganciclovir. We demonstrate that metabolic coop eration can account for this bystander effect. When HT 1080 human fibr osarcoma cells marked with a lacZ gene (LZ+5) were cocultured with HT1 080 cells transduced with a retrovirus expressing HSVtk (HT1080tk11), at a density at which the majority of cells were in contact, both HT10 80tk11 and LZ5+ cells were killed by ganciclovir. When cells were cocu ltured at a low density where the majority of cells are not in contact with one another, however, only the HT 1080tk11 cells were killed. Th is result suggests that cell contact with HT1080tk11 cells is necessar y to render the HSVtk- LZ+5 cells sensitive to ganciclovir. Because in volvement of metabolic cooperation in the killing of the LZ+5 cells wo uld require not only contact between HT 1080tk 11 and LZ+5 cells but a lso the capacity to transfer small cytotoxic molecules from the former cell to the latter, transfer of radioactive molecules between the two cell lines was assessed by autoradiography after treatment of a cocul ture with [H-3]ganciclovir. Isolated HT1080tk11 cells incorporated the labeled ganciclovir into their nuclei, whereas isolated LZ+5 cells di d not. LZ+5 cells incorporated [H-3]ganciclovir, only when in contact with HT1080tk11 cells. These findings indicate that a ganciclovir meta bolic product, presumably a phosphorylated form, can pass from HSV tk to HSV tk- cells and mediate cytotoxicity as a consequence of direct contact.