| Tumor necrosis factor-a (tumor necrosis factor-α, TNF-α) is a pleiotropic cytokine and exerts its biological activities in two forms, namely secreted TNF-α(S-TNF-α) and transmembrane TNF-α(TM-TNF-α). It has been shown in our previous studies and other laboratory's researches that TM-TNF-αhad a broader tumoricidal spectrum than S-TNF-αand was able to kill the tumor cells which are resistant to S-TNF-α. TM-TNF-αinduced mainly apoptosis while S-TNF-αcaused chiefly necrosis.In addition,TM-TNF-αis not only as a ligand that deliveries forward signal via TNF receptor to target cell but also as a receptor that transmits reverse signal to effect cells. As TM-TNF-αis the precursor of S-TNF-αand their only one difference in the structure is that TM-TNF-αhas an unusual long signal peptide (SP) consisting of 76 amino acids. This signal peptide not only anchors TM-TNF-αon cell surface membran, but also is closely related to the transmission of reverse signal via TM-TNF-α. Our previous work had confirmed that tumor cells were able to resistant apoptosis through reverse signal mediated by TM-TNF-α. Therefore, to exclude the interference of the forward signaling of TM-TNF-α, we had constructed the human breast tumor cell line expression TNF-SP and investigated the biological functional of TNF-SP on tumor cells, which provided an useful tool to further research the effect of reverse signal mediated by TM-TNF-αon tumor growth . The main results as follows:1. Construction of human breast tumor cells with expression of high levers of TNF-SPThe recombinant pIRES2-EGFP/TNF-SP, which contained human TNF-SP cDNA, and the vector pIRES2-EGFP were transduced into a human breast tumor cell line MCF-7 with Lipofectamine 2000 respectively. Cloned cell lines were obtained after G418 resistance selection. The positive cell clone, which expressed high level TNF-SP protein were selected by FACS analysis. It was found by western blotting assay that around 10kD, an apparent band was seen in the positive cell clone. No evident same band was observed in otherwise cell groups. These findings indicate that a eukaryotic cell line highly expressing TNF-SP has successfully been constructed.2. Effect of reverse signal via TNF-SP on tumor growth(1) MCF7 cells expressed high level TNF-SP can resiste the cytotoxicity of S-TNF-αThe results of MTT showed that MCF7 cells transfected with TNF-SP were resistant against the cytotoxicity of S-TNF-α(motality 3.57%, P<0.01), while transfected with pIRES2-EGFP MCF7 cells and no-transfected MCF7 cells were still sensitive to cytotoxic effect of S-TNF-α(mortality 22.45%,22.24%), suggesting that transfected with TNF-SP can make the TNF-sensitive cells become the TNF-resistant cells.(2) TNF-SP can active NF-κB by reverse signal, resulting in MCF7 cells resisting to the cytotoxicity of S-TNF-αThe results from ELISA revealed that the NF-κB activity level of TNF-SP/MCF7 cells was higher than that of the other two cells, and it significantly increased after stimulated by S-TNF-α. In addition, pre-treated with NF-κB inhibitor PDTC could inhibit the NF-κB activity of TNF-SP/MCF7cells and restore efficiently the sensibility of to S-TNF-α( the death rate increased to 36.7%, P<0.01), and it also could increase the sensitivity of the control cells to the cytotoxic effect of S-TNF-α.(3) TNF-SP can promot degradation of IκB-αby reverse signal The results of Western blotting showed that the intracellular IκB-αlevel of MCF7 transfected with TNF-SP cells was lower than that of the two kinds of control cells (control vector transfected MCF7 cells and no-transfected MCF7 cells), and the TNF-SP/MCF7 cells were more sensitive to stimulation of S-TNF-αand its level of intracellular IκB-αdecreased more significantly than the two kinds of control cells.(4) TNF-SP can increase the production of NO by reverse signal It was found that transfection with TNF-SP was capable of enhancing NO production in MCF7 cells by 50.68% (p<0.01) than the control cells.In summary, this study had successfully constructed a eukaryotic cell line highly expressing TNF-SP (TNF-SP /MCF7 cells), and demonstrated that transfected with TNF-SP gene can make MCF7 cells resiste to the cytotoxicity of TNF-αby activating NF-κB and increasing NO production, suggested that TNF-SP may be involved in the reverse signaling of TM-TNF-αvia its active modif to induce NF-κB activity increase which promoting tumor cells survival. So the TNF-SP/MCF7 cell line can provide an useful tool to further research the reverse signaling via TM-TNF-α. |
