|Budget Amount *help
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1997: ¥2,300,000 (Direct Cost: ¥2,300,000)
This experiment was conducted to develop a new method of gene therapy for head and neck cancer patients. First, we attempted to introduce a suicidal gene, HSV-tk, in an EB virus-plasmid vector (pEBETA3-CAG). When we transfected HSG cells or TYS cells, both are salivary gland cancer cells, with pEBETA 3-CAG-HSV-tk in vitro by electroporation or liposome complex method, extremely high expression of the transfected gene was observed in both HSG cells and TYS cells. However, we failed to introduce these EBETA-plasmid vector into the nude mouse tumors formed by HSG cells. Then we examined the in vivo transfection efficiency using a common plasmid vector, pGL3-control containing luciferase gene as a reporter driven by SV40 early promoter. HSG nude mouse tumor was transfected by pGL3-control in several conditions for electroporation. Two days after transfection, tumors were excised from mice, and the tissue extracts were analysed for luciferase activity. It was found that transfection of HSG nude mouse tumors with 5 mug of the plasmid by per-cutaneous electric-shock at 1 kv, 99 musec. for 8 times square pulse is the most effective condition tested for in vivo electroporation. In order to examine the localization of the transfected gene products in the cells, we used pEGFP-C3 plasmid, containing green fluorescent protein (GFP) driven by CMV-IE promoter. Two days after transfection, tumors were excised from mice, and frozen sections were prepared. We obserbed the expression of GFP fluorescent in some of the cells in the tumors under fluorescent microscopy.
We succeed to introduce plasmid vector into nude tumors by electroporation. These results suggests that these method can be applied for human head and neck cancer gene-therapy, if the transfection efficiency will be improved.