Experimental Study Of Viral Vector-mediated HyTK Gene Transfer For The Treatment Of Malignant Melanoma

Transfer of Herpes simplex virus thymidine kinase gene(HSV-tk) into tumor cells followed by ganciclovir(GCV)administration,will provide selective tumor cell killing. The machanism is as follows: GCV, a nucleoside analogue, becomes cytotoxic after being converted to its triphosphorylated form through phosphorylation by HSV thymidine kinase and host cellular kinases. The triphosphorylated GCV interferes with DNA synthesis in the replicating cells, and causes cell death. In addition, HSV-tk gene negtive tumor cells in close proximity to HSV-tk-positive tumor cells become GCV-sensitive through a phenomenon described as the "bystander effect." This effect makes it possible to eliminate all tumor cells despite only a fraction of them expressing HSV1 -tk gene.In this study, A fusion gene encoding both HSV1-TK and hygromycin phosphotransferase was used as the therapeutic gene.The retroviral vector LNCHyTK containing HyTK gene was constructed and transduced into packaging cell line PA317.Virus producing cells ( VPC ) were obtained,and the vector containing supernatant harvested. The murine melanoma cell line B16 was chosen as a major target for study of the "killing" effect mediated by retroviral-HyTK/GCV system in vitro and in vivo. The in vitro cytotoxic effect of HyTK/GCV system on other tumor cell lines including human cervical carcinoma cells HeLa and SiHa, as well as human liver cancer cells H7402 was also investigated.The results showed that retrovirus-mediated HyTK gene transfer to B16 cells could confer them high sensitivity to GCV(>100-1000 fold, compared with the parental B16 cells),and exhibit the bystander effect. Similar results were obtained from experiments with HeLa and H7402 cells. The HyTK gene transferred SiHa cells,however, showed a relatively low sensitivity to GCV and gave no effective bystander effect.