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Gap Junctions Promote the Bystander Effect of Herpes Simplex Virus Thymidine Kinase in Vivo¹

Departments of Medicine [M. S. D., B. B., G. G.], Clinical Immunology, Microbiology, Pathology, and Infection [M. R. A., C. I. E. S., B. C.], Gene Therapy Research Center, Clinical Research Center [C. I. E. S.], Huddinge Hospital, Karolinska Institute, S-14186 Huddinge, Sweden; Center for BioTechnology, the Department of Biosciences [M. S. D., C. I. E. S.], Karolinska Institute, NOVUM, S-14157 Huddinge, Sweden; and Departments of Physiology [G. M. K.] and Anatomy and Cell Biology [C. C. G. N.], The University of Western Ontario, London, Ontario, N6A 5C1 Canada

Transfer of the herpes simplex thymidine kinase gene (HSVtk) into tumor cells followed by the administration of ganciclovir (GCV) provides a potential strategy for the treatment of some malignancies. During GCV treatment, not only the cells that express the HSVtk gene are killed but also frequently neighboring tumor cells that are not genetically altered. This has been called the "bystander effect." Although the mechanism of the bystander effect in vivo remains elusive, our results suggest that gap junction formation between neighboring cells is an important contributing factor.

The C6 rat glioma cell line, which exhibits a low level of intercellular communication by gap junctions and connexin43 (Cx43)-transfected clones of this cell line forming gap junctions from a moderate level (Cx43-12 and Cx43-14) to a high level (Cx43-13), were transduced with HSVtk. Transduced and nontransduced cells were mixed in various concentrations and then cultured in vitro or injected s.c. into C.B-17/SCID-beige mice followed by i.p. injections of GCV. Cx43-transfected clones showed a significant increase of the bystander effect compared with the less coupled C6 parental cell line. In 11 of 12 mice injected with cells of Cx43-transfected clones, no tumors were seen at the inoculation site when a mixture of 50% HSVtk-negative and HSVtk-positive cells was used. Moreover, in mice injected with cells of clone Cx43-13, which exhibits the highest intercellular communication, tumors were frequently undetectable at the inoculation site when using mixtures of 75% HSVtk-negative and 25% HSVtk-positive cells, and even mixtures containing 5% HSVtk-positive cells of Cx43-transfected clones showed tumor size reduction. All animals in control groups (n = 26) developed large tumors at every injection site. These results demonstrate that gap junctions are an important component in mediating the bystander effect in vivo.

¹ This study was supported by grants from the Swedish Medical Research Council, the Swedish Cancer Society, the BIOMED Shared Cost Grant BMH4-CT96-1436, and the Cancer Research Society, Inc.

² To whom requests for reprints should be addressed, at Karolinska Institute, Hematology Laboratory, KFC, NOVUM, S-14186, Huddinge, Sweden. Phone: 46 8 7465066; Fax: 46 8 7465210; E-mail: Sirac.Dilber@medhs.ki.se.

Received 10/23/96. Accepted 2/17/97.

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