Inhibition of Tumor Growth by Direct Intratumoral Gene Transfer of Herpes Simplex Virus Thymidine Kinase Gene with DNA–Liposome Complexes

To establish the expression of the herpes simplex virus thymidine kinase (HSV-TK) gene in tumor cells, we analyzed the promoter function of the SV40 promoter and the nucleotide sequence (CACGTG) to which Myc-Max heterodimers (Myc/Max) were capable of binding in four kinds of cell lines: COLO320 DM, A-431, KF, and Nakajima. When luciferase reporter plasmid under the control of SV40 promoter was transfected into tumor cells in vitro, a high level of luciferase activity was observed in all kinds of cell lines. However, by transfection of the luciferase gene promoted by the Myc/Max binding sequence, accelerated luciferase expression was observed in COLO320 DM and A-431 cells with high expression of c-myc, but not in KF and Nakajima cells, which showed low expression of c-myc. The repeated transfection of the liposome-conjugated HSV-TK gene regulated by the SV40 promoter and cultivation in 100 μg/ml of aciclovir for 5 days in vitro demonstrated growth inhibition for all four kinds of cell lines. However, cell toxicity was observed only in COLO320 DM and A-431 cells when the HSV-TK gene promoted by the Myc/Max binding sequence was introduced. (The survival rate to 100 μg/ml of aciclovir concentration in COLO320 DM, A-431, KF, and Nakajima cells was 59%, 53%, 74%, and 79%, respectively.) In vivo direct injection of the liposome-conjugated HSV-TK gene regulated by the SV40 promoter into established tumors and aciclovir administration for 10 days into the mice resulted in significant tumor volume reduction in three tested tumor cells. However, injection of the HSV-TK gene promoted by the Myc/Max binding sequence and aciclovir administration into mice could achieve significant tumor regression only in COLO320 DM and A-431 cells. These results suggest that gene therapy using the HSV-TK gene promoted by the Myc/Max binding sequence can be an attractive approach for treatment against tumor cells expressing high levels of c-myc. By transducing the herpes simplex virus thymidine kinase (HSV-TK) gene into tumor cells with the administration of aciclovir both in vitro and in vivo, cell toxicity was observed specifically in tumor cells expressing high levels of c-myc by the Myc/Max binding sequence directing the HSV-TK gene, while the HSV-TK gene directed by the SV40 promoter killed all tumor cells. This study demonstrates that the suicide gene could be expressed selectively in tumor cells and suggests that gene therapy targeting for cancer cells might be possible if the gene is regulated by an appropriate promoter specific for tumor cells.