The Regulation Of Expression And Function Of Membrane TRAIL And Soluble TRAIL By Metalloproteases Inhibitors

Objective: To study the regulation of expression and function of membrane TRAIL and its soluble form by proteases inhibitors. Methods: FMU-TRAIL3 mAbs were purified by caprylic acid method and identified by SDS-PAGE. The recoganition of mAbs to mTRAIL on different cells was confirmed by indirect fluorescent staining and flow cytometry analysis (FCM). Its recognition to recombinant TRAIL (rTRAIL) and native TRAIL in cell lysate was identified by immune precipitation (IP) and western blot assay. The neutralizing function of FMU-TRAIL3 mAbs was investigated by cell apoptosis detection and ⁵¹Cr release assay.(2) The indirect fluorescent staining and FCM were also used to study the expression of mTRAIL on Jurkat, Daudi and Colo205 cells treated by 8 proteases inhibitors respectively. The expression of ADAM10 and ADAM17 gene in the 3 cell lines were detected by RT-PCR.(3) Jurkat cells were stimulated by PMA or PHA for indicated time periods.In some experiments , metalloproteases inhibitor 1,10-phenanlhroline and ADAM inhibitor TAPI-1 were added. The expression of mTRAIL was determined by indirect fluorescent staining and FCM., and the secretion of sTRAIL was detected by ELISA kit The same methods were also used to study mTRAIL expression and sTRAIL secretion of Daudi cells treated by IFN-a and metalloproteases inhibitor. The cytotoxicity of mTRAIL and sTRAIL was determined by ⁵¹Cr release assay and neutralizing monoclonal antibody blocking assay. The molecular weight" (MW) of sTRAIL was identified by IP and western blot assay. (4) Colo 205 cells were stimulated by 250u/ml EFN-α and cultured for indicated time periods .The expression of mTRAIL , DR4 and DR5 were also measured by indirect fluorescent staining and FCM. ,and caspase-8 activity was detected by using a FLICE/casepase -8 colorimetric kit Annexin V-FITC staining kit was used to study rTRAIL-induced apoptosis in Cok>205 cells .The same kit was also used to detect the self-induced apoptosis in Colo 205 cells treated by IFN-α or IFN-α plus TAPI-1. FMU-TRAIL3 mAb was added in some experiments. Results:(l) FMU-TRAIL3 mAb can recognize mTRAIL, rTRAIL and native TRAIL in cell lyslate .The specific lysis and apoptosis of Raji cells induced by 10ng/ml rTRAIL were 28.4% and 14.3% respectively .Whereas the addition of FMU-TRAIL3 mAb could reduce them to 8.5% and 5.9% respectively.Therefor the inhibition rate of FMU-TRAEL3 mAb to the specific lysis and apoptosis were 70.0% and 58.7% respectively.(2)The expression level of mTRAIL on resting Jurkat, Daudi and Colo205 cells were 17.7%,33.6% and 23.4%. However 1.10-phenanthroline could enhanced them to 56.7%,67.2% and 61.4% remarkbly.TAPI-1 also increased the expression of mTRAIL to 44.4%,44.1% and 47.3% evidently, while other inhibitors have no effect on mTRAIL. expression. Furthermore both ADAM10 and ADAM17 geneexpression could be detected in the 3 cell lines. Finally, the metalloproteases inhibitors regulated the mTRAIL expression on resting cells in a dose and time dependent pattern.(3) The level of sTRAIL in the supernatant PMA-stimulated Jurkat cells reached the peak after Jurkat cells were stimulated for 48 h(1997±39pg/ml).Whereas the addition of 1,10-phenanhroline and TAPI-1 reduced sTRAIL level to 1443±45pg/ml and 1566±29 pg/ml respectively. The specific lysis induced by the 1/2 diluted supernatant (Jurkat cells treated by PMA, PMA plus 1,10-phenanhroline or PMA plus TAPI-l)were 68.6%,46.6% and 55.4%, and the cell lysis could be blocked by FMU-TRAIL3 mAb by 50.4%, 62.2% and 26.7% respectively. The expression level of TRAIL on Jurkat cells treated by PMA, PMA plus 1,10-phenanthroline or PMA plus TAPI-1 for 60 h were 41.2%>67.3% and 44.7%. When the ET ratio was 200:1, the specific lysis of Raji cells induced by 3 groups of cells were 42.4 %, 71.5 % and 57.3%. However, the addition of FMU-TRAIL3 mAb could blocked specific lisis induced by the 3 groups of cells by 59.7%, 51.8% and 64.9 % respectively.Therefor, those two inhibitors regulated the function of TRAIL by adjusting the sTRAIL secretion or mTRAIL expression.. It was found that the actived Cells could released two kinds of sTRAIL with MW of 33kDa and 19kDa In the presence of 1,10-phenanthroline, only the secretion of 19kDa sTRAIL was blocked.(4)The expression of DR4 and DR5 of FN-α-stimulate Colo205 cells reached the peak after stimulating for 60 h, while the activity of Caspase-8 was relatively high in Colo205 cells stimulated by IFN-α for 24h or 36h..Compared with that of resting cells (7.6%), the apoptosis rate induced by rTRAIL to IFN-α-stirnulated for 36h Colo205 cells and 60h were 22.3% and 17.6%. IFN-α combined with TAPI-1 was also more effective in up-regulating mTRAIL expression and apoptosis of Colo 205 cells induced by themselves than IFN-a only.