Inhibitory effect of anti-fas ribozyme on apoptosis of mouse T cells.
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).


CONCLUSIONS
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.
Zhang
Min
M
Department of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, P.R.China. zhangmin35@yahoo.com.cn
Chen
Zhi-Chao
ZC
Tian
Lei
L
Liu
Fang
F
Liu
Ling-Bo
LB
You
Yong
Y
Zou
Ping
P
D000818:Animals; D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002478:Cells, Cultured; D008223:Lymphoma / Q000378:metabolism* / Q000473:pathology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016337:RNA, Catalytic / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013601:T-Lymphocytes / Q000166:cytology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014162:Transfection; D019014:fas Receptor / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
D016337:RNA, Catalytic; D012333:RNA, Messenger; D019014:fas Receptor; C427204:hammerhead ribozyme