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 < or =4 microM. 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.
Herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) gene therapy can induce apoptosis in tumor cells that are normally resistant to this type of cell death, although the cellular mechanisms by which this occurs remain to be elucidated. Human colon tumor cell lines expressing HSV-TK were treated with GCV or four other inducers of apoptosis: butyrate, camptothecin (CPT), Taxol (paclitaxel), or 7-hydroxystaurosporine (UCN-01). Over a 2-4 day treatment period with GCV or the other four drugs, protein levels of the apoptosis agonist Bak increased 1.5-to 3-fold, whereas a corresponding decrease in the levels of the apoptosis antagonist, Bcl-X L , was observed in butyrate-, CPT-, and 7-hydroxystaurosporine (UCN-01)-treated cells. GCV and paclitaxel treatments resulted in increased levels of Bcl-X L . In two-drug combinations with GCV plus one of the four other drugs, increased tumor cell killing was found with GCV plus UCN-01 or with some GCV/butyrate combinations; the other two tested combinations were largely antagonistic. The GCV/UCN-01 and GCV/butyrate combinations resulted in increased Bak and decreased Bcl-X L protein levels, while the GCV/CPT and GCV/paclitaxel combinations resulted in increased levels of both proteins. The results highlight the potential for new combination therapies of HSV-TK/GCV and chemotherapeutic drugs that result in increased tumor cell apoptosis for future treatments of colon cancer.
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