MECHANISMS FOR GANCICLOVIR RESISTANCE IN GASTROINTESTINAL TUMOR-CELLSTRANSDUCED WITH A RETROVIRAL VECTOR CONTAINING THE HERPES-SIMPLEX VIRUS THYMIDINE KINASE GENE

Transfer of the herpes simplex thymidine kinase (HSV-TK) gene into tum or cells confers sensitivity to the cells to the viral drug ganciclovi r (GCV), Although the efficacy of the HSV-TK/GCV approach is well stud ied, the mechanisms for the resistance of HSV-TK-transduced tumor cell s to GCV are poorly understood, Here, we examined the mechanisms for G CV resistance in HSV-TK-transduced gastrointestinal (GI) cell lines, O ur results show that GCV sensitivities vary in vitro and in vivo among the different HSV-TK-transduced GI tumor cell lines, GCV-resistant co lonies were isolated from several different HSV-TK-transduced GI tumor cell lines after 14 days of GCV treatment. Characterization of GCV-re sistant colonies demonstrated that the HSV-TK gene was either partiall y 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 wa s 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 a bsent in the GCV-resistant, HSV-TK-transduced cells, Transfection of t he resistant cells with an adenoviral vector containing a HSV-TK gene restored sensitivity to GCV, The presence of GCV-resistant cells was o nly demonstrable in GI tumor cell lines that also demonstrated a poor bystander effect, Our results suggest that GCV resistance found in tum or cells transduced with a retroviral HSV-TK gene is due to the lack o f 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 expres s the TK protein are probably killed by the bystander effect because G CV-resistant tumor cells were not found in these cell lines, GCV-resis tant tumor cells were found only in tumor cell lines with a poor bysta nder effect, by which, presumably, the transduced tumor cells lacking a functional TK gene were not killed by the bystander killing effect.