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ISSN
1672-9145
Acta Biochim Biophys Sin
2004, 36(8): 559–565
CN 31-1940/Q
Inhibiting Apoptosis of CTLL-2 Cells to Enhance Their GVL Effects
via Anti-Fas Ribozyme
Min ZHANG, Fang LIU, Lin-Bo LIU, Yong YOU, Zhi-Chao CHEN, and Ping ZOU*
Institute of Hematology, Union Hospital, Tongji Medical College,
Huazhong University of Science & Technology, Wuhan 430022, China
Abstract To investigate the inhibition role of anti-Fas hammerhead ribozyme
on fas expression and Fas-mediated apoptosis of CTL cell line CTLL-2
cells, the cDNA of an anti-Fas hammerhead ribozyme was synthesized, its
expression plasmid was constructed and transfected into CTLL-2 cells by
electroporation. fas expression of CTLL-2 cells was detected by RT-PCR
and Western blot. CTLL-2 cell viability was measured using MTT assay when
co-cultured with mouse T cell leukemia cell line EL4 cells that highly
expressed Fas ligand (FasL). Meanwhile, caspase-3 proteolytic activity was
detected, and cell apoptosis was measured by flow cytometry and Hochest-PI
double staining. Killing activity of CTLL-2 cells was detected by lactate
dehydrogenase (LDH) releasing assay in vitro. Results showed that the
expression of both Fas mRNA and protein in CTLL-2 cells were decreased after
transfection of anti-Fas ribozyme. Compared with mock-transfected group and
mutant ribozyme-transfected group, viability of CTLL-2 cells co-cultured with
EL4 cells was increased significantly and cells killing activity was enhanced
after transfected with anti-Fas ribozyme, while the caspase-3 activity and
apoptosis rate was significantly decreased. The results demonstrated anti-Fas
ribozyme could efficiently cleave Fas and inhibit Fas-mediated apoptosis of
CTLL-2 cells to improve their viability. Our study made a basis for enhancing
CTLL-2 cells anti-leukemia effect in DLI.
Key words Hammerhead ribozyme; anti-Fas; CTLL-2 cells; apoptosis; tumor
immunity
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Received: April 15, 2004 Accepted: June 28, 2004
This work was supported by a grant from the National Natural Science
Foundation of China (No. 30240022)
*Corresponding author: Tel, 86-27-85726880; E-mail, [email protected]