Lack of Bystander Killing in Herpes Simplex Virus Thymidine Kinase-Transduced Colon Cell Lines Due to Deficient Connexin43 Gap Junction Formation

Abstract

The efficacy of herpes simplex virus thymidine kinase (HSV-TK) gene therapy for colorectal carcinoma has been investigated in an in vitro system. The magnitude and the mechanism of the HSV-TK bystander effect was determined in three human colon tumor cell lines: HCT-116, HCT-8, and HT-29. Each HSV-TK(+) cell line was generated by stable transduction with a bicistronic retroviral vector containing the HSV-TK and neomycin resistance (neo) genes; each exhibited an IC₅₀ for GCV of ≤4 μM. When GCV was added to HSV-TK(+) cells mixed with parental cells or known bystander-positive cell lines, no bystander killing was evident in the HT-29 or HCT-8 cells. Western blots detected the expression of the gap junction protein connexin43 (Cx43) in HCT-8 and HT-29 cells; however, immunolocalization studies indicated predominantly cytoplasmic staining of Cx43 and no cell surface staining in these cell lines. Stable transfection of HCT-8 and HT-29 cells with Cx43 resulted in increased levels of Cx43 expression with the same subcellular distribution as before, yet there was again no apparent bystander killing. In contrast, Cx43 expression was localized to the cell surface in the bystander-positive colon tumor cell line HCT-116. These results demonstrate that expression and proper surface localization of Cx43 gap junctions are necessary components of the bystander effect in human colon tumor cells. They also indicate that future combination gene therapy approaches using coexpression of HSV-TK and Cx43 genes may not be applicable to all tumor systems.

Overview summary

We investigated the use of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) suicide gene therapy for the treatment of colorectal carcinoma. In spite of expression of connexin43, two colon tumor cell lines, HT-29 and HCT-8, did not exhibit bystander effect killing in comparison with the bystander-positive HCT-116 human colon tumor cell line. We demonstrate that the absence of bystander effect killing in these tumor cell lines is correlated with the lack of functional connexin43 gap junctions at the cell surface.