The “Bystander Effect”: Association of U-87 Cell Death with Ganciclovir-Mediated Apoptosis of Nearby Cells and Lack of Effect in Athymic Mice

Abstract

Cells expressing the herpes simplex-thymidine kinase (HS-TK) gene as a consequence of retroviral transduction, as well as TK-negative (TK¯) bystander cells, can be killed by treatment with ganciclovir (GCV). In vitro, this “bystander effect,” has been attributed to metabolic cooperation through gap junctions or to the uptake of apoptotic vesicles. We show that GCV treatment kills TK-negative U-87 glioma cells cocultured with cells that express TK (TK⁺) but that have lost the capacity for releasing retroviral particles. A photometric enzyme immunoassay identifies histone-associated DNA fragments, typical of apoptosis, in the cytosol of GCV-treated TK⁺ cells, and apoptotic features are also demonstrated by ultrastructural studies. Northern blot analysis and the reverse transcription polymerase chain reaction (PCR) show that connexin 43, a major constituent of gap junctions, is expressed in TK⁺ and U-87 cells. The size of U-87 tumors in nude mice subsequently injected with TK⁺ cells and GCV is not significantly different than in untreated animals; whereas, after injecting 1:1 mixtures of U-87 and TK⁺ cells, GCV treatment only causes a temporary regression of tumor growth. On the contrary, when the injected mixtures contain PA317.STK.SBA (a retroviral producer cell line that can transduce efficiently the HS-TK gene) and U-87 cells, tumors are destroyed effectively by GCV treatment. Thus, an experimental setting in which U-87 gliomas are matched with cells that are able to express, but not to transduce, the HS-TK gene indicates that the bystander effect kills U-87 cells in vitro by mechanisms associated with apoptotic death. In vivo, this effect is not sufficient to restrain the tumor growth taking place in immunodeficient animals.

Overview summary

This paper participates in the debate on the nature of the bystander effect, the phenomenon by which tumor cells that surround but have not been transduced by herpes simplex-thymidine kinase retroviral producer cells die when treated by the nucleoside analogue ganciclovir (GCV). We suggest that the release of vesicles during GCV-mediated apoptotic cell death is a major constituent of the bystander effect. Gap junctions may have a role in this process, even if the expression of proteins constituting their subunits, such as connexin 43, is generally low in tumors. Experiments in athymic mice confirm that the bystander effect facilitates tumor regression, but that retroviral transduction remains of critical relevance in making tumor eradication efficient.