BYSTANDER KILLING OF CANCER-CELLS BY HERPES-SIMPLEX VIRUS THYMIDINE KINASE GENE IS MEDIATED BY CONNEXINS

In gene therapy to treat cancer, typically only a fraction of the tumo r cells can be successfully transfected with a gene. However, in the c ase of brain tumor therapy with the thymidine kinase gene from herpes simplex virus (HSV-tk), not only the cells transfected with the gene b ut also neighboring others can be killed in the presence of ganciclovi r. Such a ''bystander'' effect is reminiscent of our previous observat ion that the effect of certain therapeutic agents may be enhanced by t heir diffusion through gap junctional intercellular communication (GJI C). Herein, we present the evidence, from in vitro studies, that gap j unctions could indeed be responsible for such a gene therapy bystander effect. We used HeLa cells for this purpose, since they show very lit tle, if any, ability to communicate through gap junctions. When HeLa c ells were transfected with HSV-tk gene and cocultured with nontransfec ted cells, only HSV-tk-transfected HeLa cells (tk(+)) were killed by g anciclovir. However, when HeLa cells transfected,vith a gene encoding for the gap junction protein, connexin 43 (Cx43), were used, not only tk(+) cells, but also tk(-) cells were killed, presumably due to the t ransfer, via Cx43-mediated GJIC, of toxic ganciclovir molecules phosph orylated by HSV-tk to the tk(-) cells. Such bystander effect was not o bserved when tk(+) and tk(-) cells were cocultured without direct cell -cell contact between those two types of cells. Thus, our results give strong evidence that the bystander effect seen in HSV-tk gene therapy may be due to Cx-mediated GJIC.