Gene therapy for human ovarian cancer cells using efficient expression of fas gene combined with γδT cells

Abstract

Low tissue specificity and efficiency of exogenous gene expression are the two major obstacles in tumor-targeted gene therapy. The Fas cell surface death receptor (Fas)/Fas ligand pathway is one of the primary pathways responsible for the regulation of cell apoptosis. The aim of the present study was to explore whether the regulation of tumor specific promoters and a two-step transcriptional amplification system (TSTA) assured efficient, targeted expression of their downstream Fas gene in human ovarian cancer cells, and to assess the killing effect of γδT cells on these cells with high Fas expression. Three shuttle plasmids containing different control elements of the human telomerase reverse transcriptase (hTERT) promoter and/or TSTA were constructed and packaged into adenovirus 5 (Ad5) vectors for the expression of exogenous Fas gene. The human ovarian cancer cell line SKOV3 a nd a cont r ol human embryonic lung fibroblast cell line were transfected with Ad5-hTERT-Fas or Ad5-hTERT-TSTA-Fas. Fas mRNA and protein expression were examined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. γδT lymphocytes were isolated, cultured and mixed at different ratios with SKOV3 cells with Fas expression in order to assess the killing effect of γδT cells. hTERT promoter induced the specific expression of FAS gene in SKOV3 cells, and the TSTA strategy increased FAS expression by 14.2-fold. The killing effect of γδT cells increased with the expression level of Fas and the effector-target cell ratio. The killing rate for SKOV3 cells with high FAS expression was 72.5% at an effector-target cell ratio of 40:1. The regulators of hTERT promoter and TSTA assure the efficient and targeted expression of their downstream Fas gene in SKOV3 cells. The killing effect of γδT cells for ovarian cancer cells with relatively high Fas expression was improved.