Background & objective: Tumor cells can express FasL, and induce apoptosis of activated T cells expressing Fas, which is so called "Fas counterattack". This study was to investigate inhibitory effect of anti-Fas ribozyme on apoptosis of mouse T cells, and explore a new way to enhance antitumor ability of T cells.
Methods: A hammerhead ribozyme targeting Fas mRNA was synthesized, its in vitro cleavage reaction was performed. Anti-Fas ribozyme was transfected into mouse spleen T cells by electroperation (ribozyme-transfected group), empty control and mock-transfected groups were set up. Fas expression on T cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. After treatment of anti-Fas antibody (JO(2)), cell apoptosis was measured by flow cytometry, proliferation of T cells was measured by MTT assay. In vitro killing activity of cytotoxic T lymphocytes (CTLs) was detected by lactate dehydrogenase (LDH) releasing assay.
Results: Anti-Fas ribozyme cleaved Fas mRNA efficiently with a rate of 60%. mRNA level of Fas was significantly lower on ribozyme-transfected cells than on control and mock-transfected cells (0.4 vs. 1.1, and 1.0, P < 0.01)u its protein level was according to this result. After treatment of JO(2), cell apoptosis rate was significantly lower in ribozyme-transfected group than in the rest 2 groups (35% vs. 86%, and 87%, P < 0.01), but cell proliferation rate was significantly higher in ribozyme-transfected group than in the rest 2 groups (208% vs. 100%, and 98%, P < 0.01). In vitro killing activity of CTLs was significantly stronger in ribozyme-transfected group than in the rest 2 groups (67% vs. 32%, and 31%, P < 0.01).
Conclusion: Anti-Fas ribozyme can remarkably suppress Fas expression on mouse activated spleen T cells, and protect T cells from Fas-mediated apoptosis, which may enhance antitumor ability of T cells.