The Molecular Mechanisms Of A Novel Agonistic Anti-human DR5 Monoclonal Antibody Inducing Apoptosis In Leukemia Cell Lines

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily with the ability to induce apoptosis in a wide variety of transformed cell lines of diverse origin. At least five receptors for TRAIL have been identified so far. Two of them, DR4 (TRAIL-R1) and DR5 (TRAIL-R2),are capable of transducing apoptosis signal,whereas the other three, DcR1 (TRAIL-R3), DcR2 (TRAIL-R4) and osteoprotegerin (OPG), serve as decoy receptors to block TRAIL-mediated apoptosis. DR4 and DR5 share a commom intracellular domain, called the death domain (DD), which is indispensable for initiation of the intracellular signaling cascade leading to cell death. TRAIL has been known to induce apoptosis in a variety of tumor cells and some virally infected cells but not in most normal cells. Monoclonal antibodies (mAbs) against TRAIL receptors with tumoricidal activity are also potential candidates for cancer therapy. There are a number of agonistic mAbs against human DR4 or DR5 reported in previous studies, most of which need crosslinkers to ensure effective killing of tumor cells. In 2001, Ichikawa et al. reported a mouse anti-DR5 mAb, TRA-8, which showed strong tumoricidal activity in the absence of cross-linking and had no hepatocyte toxicity. TRA-8 competes with TRAIL for binding to DR5 and almost entirely mimics the apoptosis-inducing mechanism of TRAIL,and the authors believed that TRA-8 was not sufficient to trigger apoptosis in normal hepatocytes. However, a recent study showed that at least some anti-DR5 and anti-DR4 mAbs did induce human hepatocytes apoptosis. Therefore, we can not draw a definite conclusion on the hepatocyte toxicity of soluble TRAIL or mAbs against TRAIL receptors by now. Studies on the mechanism of anti-DR5 mAbs will help us to understand the complicated signal pathways mediated by DR5.Objectives: To observe the effect of a novel anti-human DR5 monoclonal antibody (mDRA-6) on the apoptosis of human leukemia Jurkat,U937,HL-60 and Raji cells and its molecular mechanisms.Methods:Anti-human DR5 monoclonal antibody designated as mDRA-6, was prepared by BALB/c mouse immunization with sDR5,and then mouse spleen cells fused with myeloma NS-1 cells at the presence of 50% PEG (polyethylene glycol). Binding activity of mDRA-6 with sDR5 was determined by ELISA assay. mDRA-6 was purified by protein G affinity chromatography. Flow cytometry was used to profile the DR5 distribution on leukemic cells. Cell growth rate was detected by MTT assay, the leukemic cells image treated with mDRA-6 (10mg/L) was observed under micros cope ,and Annexin V-FITC/PI double staining quantified apoptotic outcome after mDRA-6 exposure. An indirect ELISA-based assay was established,in the ELISA -based assay, the active NF-κB in four kinds of leukemia cells after treated by mDRA-6 is captured by a double-stranded DNA probe with biotin-labeled pre-linked on multi-well plates which was labeled by avidin. Caspase 8, caspase 10, caspase 3, caspase 9 and cytochrome c was analyzed by Western blotting after mDRA-6 treatment. Furthermore, the inhibitors for Caspase 8,10,3,9 were used to block the alternatively apoptotic pathways. Mitochondria membrane potential on leukemic cells were detected by JC-1 single staining.Results:1. DR5 expression on cell membrane was distinct in different biological behavior, which were 97.474%,74.71%,58.8%,42.35% on Jurkat,U937,HL-60 and Raji cells, respectively.2. MTT analysis found that 10μg/ml mDRA-6 treated for 24h suppressed leukemia cells growth pronoucedly, Jurkat (85.12%), U937 (52.83%), HL-60 (36.48%) and Raji (30.46%) , respectively.3. Leukemic cells treated with mDRA-6 (10mg/L) exhibited typical apoptostic morphology after mDRA-6 treatment at 10μg/mL for 4 h.The apoptotic rate was 68.93%,41.95%,32.61%,25.83% for Jurkat,U937,HL-60,Raji for2 h with mDRA-6 treatment, respectively. Mitochondria membrane potential were decreased 52.35% and 51.96% for Jurkat and U937.4. Western blotting revealed that caspase-3,-9 were actived in Jurkat,U937 and HL-60 cells treated with mDRA-6,while in U937 and HL-60 cells,it is caspase-10 were activated.5. Caspase-3,-9 inhibitor partly inhibited the apoptosis of Jurkat,U937 and HL-60 cells induced by mDRA-6; the rate of cells death induced by mDRA-6 reduced77.32% (P>0.05)4.53%(P>0.05)and 8.1%(P>0.05) on Jurkat,U937,HL-60 cells respectively which pretreated with caspase-8 inhibitor . the rate of cells death induced by mDRA-6 reduced15.25%(P>0.05);69.09%(P<0.05) and77.23%(P<0.05)on Jurkat,U937,HL-60 cells respectively which pretreated with caspase-10 inhibitor.6. The activated NF-κB was appeared in Jurkat,U937 and HL-60 cells after treated by mDRA-6 for 15 min and arrived at peak for 30min,then declined.Conclusions:1. DR5 expression on Jurkat cells was higher than other leukemic cells (U937,HL-60,Raji).2.There was active NF-ΚB protein in leukemic cells after treated with mDRA-6.3. mDRA-6 is an agonistic antibody of DR5, which induces Jurkat,U937,HL-60 andRaji cells apoptosis through binding to DR5 followed by apoptotic cascades activation, i.e. caspase 8, caspase 10, caspase 3, caspase 9 and cytochrom c.In different cells, the apoptosis signal induced by mDRA-6/DR5 were transducted by different initiator caspases, caspase 8 in Jurkat, caspase 10 in U937 cells respectively.