Mechanisms for ganciclovir resistance in gastrointestinal tumor cells transduced with a retroviral vector containing the herpes simplex virus thymidine kinase gene.

Transfer of the herpes simplex thymidine kinase (HSV-TK) gene into tumor cells confers sensitivity to the cells to the viral drug ganciclovir (GCV). Although the efficacy of the HSV-TK/GCV approach is well studied, the mechanisms for the resistance of HSV-TK-transduced tumor cells to GCV are poorly understood. Here, we examined the mechanisms for GCV resistance in HSV-TK-transduced gastrointestinal (GI) cell lines. Our results show that GCV sensitivities vary in vitro and in vivo among the different HSV-TK-transduced GI tumor cell lines. GCV-resistant colonies were isolated from several different HSV-TK-transduced GI tumor cell lines after 14 days of GCV treatment. Characterization of GCV-resistant colonies demonstrated that the HSV-TK gene was either partially or completely deleted from the resistant HSV-TK-transduced cells. In the HT-29 RM and MIAPACA-2 RM cells, a 220-bp deletion of the gene was found, whereas in the HT-29 R1-R5-resistant cells, the whole TK gene was found to be absent. Immunocytochemical studies using a polyclonal antibody to the TK protein demonstrated that the HSV-TK protein was absent in the GCV-resistant, HSV-TK-transduced cells. Transfection of the resistant cells with an adenoviral vector containing a HSV-TK gene restored sensitivity to GCV. The presence of GCV-resistant cells was only demonstrable in GI tumor cell lines that also demonstrated a poor bystander effect. Our results suggest that GCV resistance found in tumor cells transduced with a retroviral HSV-TK gene is due to the lack of a functional TK protein in the tumor cells rather than any intrinsic resistance of the cells to GCV. In tumor cells with a good bystander effect, the small percentage of TK-transduced cells that do not express the TK protein are probably killed by the bystander effect because GCV-resistant tumor cells were not found in these cell lines. GCV-resistant tumor cells were found only in tumor cell lines with a poor bystander effect, by which, presumably, the transduced tumor cells lacking a functional TK gene were not killed by the bystander killing effect.