Inhibiting apoptosis of CTLL-2 cells to enhance their GVL effects via anti-Fas ribozyme.
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.
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. zhangmin35@yahoo.com.cn
Min
M
Zhang
Fang
F
Liu
Lin-Bo
LB
Liu
Yong
Y
You
Zhi-Chao
ZC
Chen
Ping
P
Zou
D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D001483:Base Sequence; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D020544:Graft vs Leukemia Effect / Q000235:genetics* / Q000276:immunology / Q000502:physiology*; D066298:In Vitro Techniques; D051379:Mice; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014162:Transfection; D019014:fas Receptor / Q000235:genetics*
D018076:DNA, Complementary; D016337:RNA, Catalytic; D012333:RNA, Messenger; D019014:fas Receptor; C427204:hammerhead ribozyme; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases