| Although the application of gene therapy has brought new hopes to the cure of cancer and other severe diseases, now available protocols are far from satisfying because of poor therapeutic efficiency or low tumor specificity. Apoptosis is a spontaneous cell death process that occurs during ontogenesis or in pathological status, while the cleavage and activation of caspases is a common downstream event in different pathways of apoptosis. Caspases exist as proenzymes in cells, and once activated they can cleave various cellular protein substrates at concensus amino acid sites, leading the cells to apoptosis. Caspase-3 is one of the most important executioners of apoptosis, and its activation causes intense and irreversible apoptosis. A specific application of caspase-3 on tumor cells may provide novel approaches for cancer gene therapy with high efficacy and low toxicity.It was previously reported that active formof caspase-3 could be acquired when the large and small subunits of caspase-3 were reversed. Here by fusing translocation domains of Pseudomonas exotoxin A (PE) N-terminus to reversed caspase-3 (revcasp-3) , we constructed 4 caspase-3 fusion proteins, as well as PE aa 253-279 truncated fusion proteins, namely "recombinant caspase-3 s" (Rcasp-3s) with or without caspase-3 recognition amino acid sequence (IETD) between PE peptide and revcasp-3. These recombinantcaspase-3s were transciently transfected into human cervical carcinoma Hela cells and other tumor cells. The transfected cells then displayed declined viabilities, and the deaths of most cells were observed through inverted microscope and fluorescent microscope. Meanwhile, the cleavage of poly (ADP-ribose) polymerase (PARP) was detected through Western blot. MTT assay indicates similar intensities of death induction of Rcasp-3s to that of revcasp-3. Revcasp-3s fused to PE aa280-364 showed slightly stronger activities than those fused to PE aa280-412, while the presence or absence of caspase-3 recognition peptide had little effect on revcasp-3 activity. We next established Rcasp-3 inducible HeLa cells, and a immediate cell death occurred following induction with ponasterone A (pon A). The order of death ratio of different Rcasp-3 s was completely identical to that in MTT assay of Rcasp-3 transciently expressing cells. HeLa cell cycles were examined following the induction of Rcasp-3 s without IETD site, and the apoptotic peaks were detected 24h post-induction. Dying cells displayed bubbly membrane, condensation of chromatin, or formation of apoptotic bodies, which are all typical features of apoptotic cells. These results proved that recombinant caspase-3 s had strong activity in inducing apoptosis of tumor cells, which suggested that the fusion of N-terminal PE peptide had hardly reduced the pro-apoptotic activity of revcasp-3, while the effect of caspase-3 recognition site between them was neglectable.In our research, novel "immunocasp-3" genes were further generated, coding fusion proteins of leading peptide, sigle-chain anti-TSA (tumor specific antigen) antibody, translocation domain of PE, and revcasp-3. The "immunocasp-3" genes were introduced into human lymphoma Jurkat cells, which still showed a normal growth curve. The expression and secretion of immunocasp-3s were verified by RT-PCR and ELISA assay. The secreted immunocasp-3 protein thus specifically recognizes HER2 overexpressed tumor cells, translocates into cytoplasma, and commits auto-cleavage between Arg 279 and Gly 280. The released recombinant caspase-3, which was alreadyproved to have comparable pro-apoptotic activity to revcasp-3, kills the cell efficiently by inducing apoptosis. The targeted pro-apoptotic activity of immunocasp-3s was confirmed by co-culture of modified Jurkat cells with HER2 possitive rumor cells. DNA ladder in genomic DNA electrophoresis indicates that the killing was due to the pro-apoptotic activity of immunocasp-3s that internalized into tumor cells.Since the immunocasp-3 gene modified lymphocytes have normal viability, they are bound to...
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