[Experimental study of inhibiting CTLL-2 cell apoptosis and enhancing cytotoxicity to Yac-1 cell by hammerhead ribozyme].
OBJECTIVE
To investigate the inhibition role of anti-Fas hammerhead ribozyme on Fas expression and Fas-mediated apoptosis in mouse cytotoxic T lymphocyte (CTL) cell line--CTLL-2 cells, and explore a novel approach to enhance the ability of T cells against leukemia in donor lymphocytes infusion (DLI).


METHODS
A hammerhead ribozyme targeting the Fas mRNA was synthesized and transfected into CTLL-2 cells by electroporation. Fas expression in CTLL-2 cells was detected by using RT-PCR, Western blot and flow cytometry, CTLL-2 cells viability was measured by MTT assay, caspase-3 proteolytic activity by caspase-3 detection kit, and cell apoptosis by flow cytometry. Killing activity of CTLL-2 was detected by LDH releasing assay in vitro.


RESULTS
Expression of Fas mRNA and protein in CTLL-2 cells was reduced to 50% after transfection with anti-Fas ribozyme. Being treated with anti-Fas antibody (JO(2)), compared with control and mock-transfected cells, viability of CTLL-2 cells transfected with anti-Fas ribozyme increased by 1-fold, caspase-3 activity and apoptosis rate of ribozyme-transfected cells decreased to 50% and 37%, respectively, and cell killing activity was enhanced by 2-fold.


CONCLUSIONS
Anti-Fas ribozyme can cleave Fas efficiently and inhibit Fas-mediated apoptosis of CTLL-2 cells, resulting in improvement of their viability.
Zhang
Min
M
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430022, China.
Liu
Fang
F
You
Yong
Y
He
Wei
W
Zou
Ping
P
Chen
Zhi-chao
ZC
Liu
Zhong-ping
ZP
Liu
Ling-bo
LB
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D014162:Transfection; D019014:fas Receptor / Q000235:genetics* / Q000378:metabolism
D016337:RNA, Catalytic; D012333:RNA, Messenger; D019014:fas Receptor; C427204:hammerhead ribozyme; D053148:Caspase 3