The role of cellular- and prodrug-associated factors in the bystander effect induced by the Varicella tester and Herpes simplex viral thymidine kinases in suicide gene therapy

To investigate the factors influencing the bystander effect - a key element in the efficacy of suicide gene therapy against cancer we compared the eff ect triggered by four extremely efficient gene/prodrug combinations, i.e., VZVtk/BVDU, the thymidine kinase of Varicella zoster virus associated with (E)-5-(2-bromovinyl)-2'-deoxyuridine; VZVtk/BVaraU, the same enzyme associa ted with (E)-5-(2-bromovinyl)-1-beta -D-arabinofuranosyluracil; HSVtk/BVDU, the association of the Herpes simplex virus thymidine kinase with BVDU; an d the classical HSVtk/GCV (ganciclovir) paradigm. The cells used, the human MDA-MB-435 breast cancer, and the rat 9L glioblastoma lines were equally s ensitive in vitro to these four associations. In both cell types, the combi nations involving pyrimidine analogues (BVDU, BVaraU) displayed a smaller b ystander killing than the combination involving the purine analogue (GCV). In addition, the bystander effect induced by all the tk/prodrug systems was reduced in MDA-MB-435 cells in comparison to 9L cells; albeit, the viral k inases were produced at a higher level in the breast cancer cells. All syst ems induced apoptotic death in the two cell types, but the MDA-MB-435 cells , deprived of connexin 43, were noncommunicating in striking contrast with the 9L cells. That functional gap junctions have to be increased in order t o improve the breast cancer cell response to suicide gene therapy was demon strated by transducing the Cx43 gene: this modification enhanced the bystan der effect associated in vitro with GCV treatment and, by itself, decreased the tumorigenicity of the untreated cells. However, the noncommunicating M DA-MB-435 cells triggered a significant bystander effect both in vitro and in vivo with the HSVtk/GCV system, showing that communication through gap j unctions is not the only mechanism involved.