In vitro and in vivo effects of retrovirus-mediated transfer of the connexin 43 gene in malignant gliomas: consequences for HSVtk/GCV anticancer gene therapy

Abstract

In tumors, gap junctional intercellular communication (GJIC) is usually down-regulated and the expression of connexins, membrane proteins constituting gap junction channels, is often low or altered. GJIC, allowing the intercellular diffusion of ganciclovir (GCV) triphosphate, is also one mediator of the ‘bystander effect’, the phenomenon by which herpes simplex virus thymidine kinase (HSVtk)-transduced, neoplastic cells kill surrounding HSVtk-negative cells when treated with GCV. We set up experiments to evaluate the effects of retrovirus-mediated in vivo gene transfer of connexin 43 in malignancies with low GJIC capacity. We found that U-87 human glioblastoma cells transfected in vitro by the human Cx43 cDNA grow signifi- cantly more slowly than control U-87 cells and lose their tumorigenicity when injected subcutaneously in nude mice. When the Cx43 gene was transduced in vitro in U-87 cells by a retroviral producer cell line (N3.2.ii, titer 1.5 × 10⁶ c.f.u./ml) in vivo results were similar. However, only when U-87 cells were co-injected with N3.2.ii cells in nude mice in a 1:5 ratio, a 50% reduction in tumor size was obtained during the first 3 weeks. Moreover the co-injection of U-87 cells with N3.2.ii and SBA cells (a retroviral producer cell line expressing the HSVtk gene), was not able to potentiate the effects of GCV administration, suggesting that Cx43 gene transfer requires more efficient vectors to increase the bystander effect in vivo.