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ISSN 1672-9145 Acta Biochim Biophys Sin 2004, 36(8): 559–565 CN 31-1940/Q

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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]