Refolding and purification of Apo2L/TRAIL produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli

As a new member of tumor necrosis factor (TNF) superfamily, TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) was produced mainly as inclusion bodies by recombinant Escherichia coli with a temperature-inducible expression system. High concentrations of both biomass (65 g dry cells l(-1)) and inactive TRAIL (4.8 g l(-1)) were obtained by applying a high-cell-density cultivation procedure. After the inclusion bodies were washed and solubilized. TRAIL refolded when at 1 mg ml(-1) by a simple pulse dilution method with a 35% yield. Renatured TRAIL was purified to electrophoretic homogeneity by one-step immobilized metal affinity chromatography. The purified TRAIL showed strong cytotoxicity activity against human pancreatic 1990 tumor cells, with ED50 about 1.6 microg ml(-1).     

High-level production of soluble tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) in high-density cultivation of recombinant Escherichia coli using a combined feeding strategy

Recombinant Escherichia coli strain C600/pBV-TRAIL (encoding for 114-281 amino acids of TRAIL's soluble fragment) produced a recombinant human tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL). Using a combined strategy of exponential feeding and pH-stat feeding, high concentrations of biomass (65 g dry wt l(-1)) and active soluble TRAIL (1.4 g l(-1)) were obtained within 30 h. The accumulation of acetate, which usually occurs during the process of high-density culture of Escherichia coli and especially in the induction stage of protein synthesis, was avoided.     

TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL.

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL-R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL-R2, by ligand-based affinity purification and subsequent molecular cloning. TRAIL-R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL-R2 contains two extracellular cysteine-rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell-surface-expressed TRAIL-R2, and TRAIL-induced apoptosis is inhibited by a TRAIL-R2-Fc fusion protein. TRAIL-R2 mRNA is widely expressed and the gene encoding TRAIL-R2 is located on human chromosome 8p22-21. Like TRAIL-R1, TRAIL-R2 engages a caspase-dependent apoptotic pathway but, in contrast to TRAIL-R1, TRAIL-R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family.     

Expression of TRAIL and TRAIL receptors in normal and malignant tissues

TRAIL, tumor necrosis factor-related apoptosis-inducing ligand, is a member of the TNF family of proteins.Tumour cells were initially found to have increased sensitivity to TRAIL compared with normal cells, raising hopes that TRAIL would prove useful as an anti-tumor agent. The production of reliable monoclonal antibodies against TRAIL and its receptors that can stain fixed specimens will allow a thorough analysis of their expression on normal and malignant tissues. Here we report the generation of monoclonal antibodies against TRAIL and its four membrane-bound receptors(TR1-4), which have been used to stain a range of normal and malignant cells, as routinely fixed specimens. Low levels of TRAIL expression were found to be limited mostly to smooth muscle in lung and spleen as well as glial cells in the cerebellum and follicular cells in the thyroid. Expression of the TRAIL decoy receptors (TR3 and 4) was not as widespread as indicated by Northern blotting, suggesting that they may be less important for the control of TRAIL cytotoxicity than previously thought. TR1 and TR2 expression increases significantly in a number of malignant tissues,but in some common malignancies their expression was low, or patchy, which may limit the therapeutic role of TRAIL.Taken together, we have a panel of monoclonal antibodies that will allow a better assessment of the normal role of TRAIL and allow assessment of biopsy material, possibly allowing the identification of tumors that may be amenable to TRAIL therapy.     

Tissue distribution of the death ligand TRAIL and its receptors.

Recombinant human (rh) TNF-related apoptosis-inducing ligand (TRAIL) harbors potential as an anticancer agent. RhTRAIL induces apoptosis via the TRAIL receptors TRAIL-R1 and TRAIL-R2 in tumors and is non-toxic to nonhuman primates. Because limited data are available about TRAIL receptor distribution, we performed an immunohistochemical (IHC) analysis of the expression of TRAIL-R1, TRAIL-R2, the anti-apoptotic TRAIL receptor TRAIL-R3, and TRAIL in normal human and chimpanzee tissues. In humans, hepatocytes stained positive for TRAIL and TRAIL receptors and bile duct epithelium for TRAIL, TRAIL-R1, and TRAIL-R3. In brains, neurons expressed TRAIL-R1, TRAIL-R2, TRAIL-R3 but no TRAIL. In kidneys, TRAIL-R3 was negative, tubuli contorti expressed TRAIL-R1, TRAIL-R2, and TRAIL, and cells in Henle's loop expressed only TRAIL-R2. Heart myocytes showed positivity for all proteins studied. In colon, TRAIL-R1, TRAIL-R2, and TRAIL were present. Germ and Leydig cells were positive for all proteins studied. Endothelium in liver, heart, kidney, and testis lacked TRAIL-R1 and TRAIL-R2. In alveolar septa and bronchial epithelium TRAIL-R2 was expressed, brain vascular endothelium expressed TRAIL-R2 and TRAIL-R3, and in heart vascular endothelium only TRAIL-R3 was present. Only a few differences were observed between human and chimpanzee liver, brain, and kidney. In contrast to human, chimpanzee bile duct epithelium lacked TRAIL, TRAIL-R1, and TRAIL-R3, lung and colon showed no TRAIL or its receptors, TRAIL-R3 was absent in germ and Leydig cells, and vascular endothelium showed only TRAIL-R2 expression in the brain. In conclusion, comparable expression of TRAIL and TRAIL receptors was observed in human and chimpanzee tissues. Lack of liver toxicity in chimpanzees after rhTRAIL administration despite TRAIL-R1 and TRAIL-R2 expression is reassuring for rhTRAIL application in humans.     

The potential of TRAIL for cancer chemotherapy

Innate and acquired resistance to chemotherapy and radiation therapy has been a major obstacle for clinical oncology. One potential adjunct to such conventional treatments is direct induction of cell death by activation of death receptor-mediated apoptosis. TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand), a recently identified member of the growing TNF superfamily, binds to its cognate death receptors DR4 and DR5 as well as decoy receptors DcR1 and DcR2. Upon binding, rapid apoptosis is enacted in a variety of human cancer cell lines independent of p53 status, but not in normal cell lines. TRAIL treatment results in significant growth suppression of TRAIL-sensitive human cancer xenografts in mice. Furthermore, combination treatment of TRAIL with genotoxic chemotherapeutic agents synergistically suppresses growth of tumor xenografts which are otherwise resistant to treatment with TRAIL or chemotherapy alone. Unlike the other death ligands TNF- or FasL, systemic administration of soluble human TRAIL does not cause toxicity in mice and non-human primates. While further studies are needed to evaluate the possible cytotoxicity of TRAIL especially for human hepatocytes, indications are increasing that TRAIL may be a novel therapeutic agent for human cancer.     

Inducing rigid local structure around the zinc-binding region by hydrophobic interactions enhances the homotrimerization and apoptotic activity of zinc-free TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), existing as homotrimer in solution, contains a unique zinc-binding site coordinated by three Cys230 residues at the tip of trimeric interface. TRAIL mutant with replacements of Cys230 with Ala (TRAIL(C230A)) negligibly formed trimeric structure and showed no apoptotic activity. Here, to elucidate the relationship between the trimeric stability and the apoptotic activity of TRAIL(C230A), we rationally designed mutations to induce homotrimerization of TRAIL(C230A) by substituting for the three residues involved in hydrogen bonding (Tyr183 and Tyr243) and putative repulsive electrostatic (Arg227) interactions at the buried trimeric interface into hydrophobic residues, like Y183F, Y243F, and R227I. The TRAIL(C230A)-derived mutants exhibited enhanced homotrimerization, but only the mutants containing R227I exhibited significant apoptosis-inducing activity in cancer cells. These results, together with the induction of rigid local structure around the zinc-binding region by R227I in TRAIL(C230A), suggest that ordered, rigid structure around the zinc-binding region is critical for the homotrimerization and apoptotic activity of TRAIL.     

CD70-restricted specific activation of TRAILR1 or TRAILR2 using scFv-targeted TRAIL mutants

To combine the CD27 stimulation inhibitory effect of blocking CD70 antibodies with an antibody-dependent cellular cytotoxicity (ADCC)-independent, cell death-inducing activity for targeting of CD70-expressing tumors, we evaluated here fusion proteins of the apoptosis-inducing TNF family member TRAIL and a single-chain variable fragment (scFv) derived from a high-affinity llama-derived anti-human CD70 antibody (lαhCD70). A fusion protein of scFv:lαhCD70 with TNC-TRAIL, a stabilized form of TRAIL, showed strongly enhanced apoptosis induction upon CD70 binding and furthermore efficiently interfered with CD70-CD27 interaction. Noteworthy, introduction of recently identified mutations that discriminate between TRAILR1 and TRAILR2 binding into the TRAIL part of scFv:lαhCD70-TNC-TRAIL resulted in TRAIL death receptor-specific fusion proteins with CD70-restricted activity.     

Acceleration of human neutrophil apoptosis by TRAIL

Neutrophil granulocytes have a short lifespan, with their survival limited by a constitutive program of apoptosis. Acceleration of neutrophil apoptosis following ligation of the Fas death receptor is well-documented and TNF-alpha also has a transient proapoptotic effect. We have studied the role of the death receptor ligand TRAIL in human neutrophils. We identified the presence of mRNAs for TRAIL, TRAIL-R2, and TRAIL-R3, and cell surface expression of TRAIL-R2 and -R3 in neutrophil populations. Neutrophil apoptosis is specifically accelerated by exposure to a leucine zipper-tagged form of TRAIL, which mimics cell surface TRAIL. Using blocking Abs to TRAIL receptors, specifically TRAIL-R2, and a TRAIL-R1:FcR fusion protein, we have excluded a role for TRAIL in regulating constitutive neutrophil apoptosis. No additional proapoptotic effect of leucine zipper TRAIL was identified following TRAIL treatment of neutrophils in the presence of gliotoxin, an inhibitor of NF-kappaB, suggesting TRAIL does not activate NF-kappaB in human neutrophils. TRAIL treatment of human neutrophils did not induce a chemotactic response. The susceptibility of neutrophils to TRAIL-mediated apoptosis suggests a role for TRAIL in the regulation of inflammation and may provide a mechanism for clearance of neutrophils from sites of inflammation.     

Expression of TRAIL receptors in human autoreactive and foreign antigen-specific T cells

Deletion of T cells due to apoptosis induction is a regulatory mechanism in the human immune system that may be impaired in autoimmune diseases such as multiple sclerosis (MS). Involvement of the apoptosis-mediating CD95/CD95 ligand system in MS has been demonstrated. Here, we report that (auto)antigen-specific human T cells are not killed in vitro by soluble TNF-related apoptosis-inducing ligand (TRAIL) although expressing death-inducing receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2. Apoptosis was assessed by caspase activation and DNA fragmentation, receptor expression was detected by RT - PCR and flow cytometry. The (auto)antigen-specific T cells were also resistant to specific TRAIL-R1/TRAIL-R2-directed induction of apoptosis, indicating that coexpression of the truncated TRAIL-R3 and TRAIL-R4 in these T cells is not responsible for the observed resistance. Upon stimulation, levels of death-inducing TRAIL receptors decreased whereas TRAIL was up-regulated on the cell surface. In contrast to CD95, the role of TRAIL receptors in MS might not involve regulation of T cell vulnerability.     

Association of TRAIL and Its Receptors with Large-Artery Atherosclerotic Stroke

Objective

To investigate the association of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors, osteoprotegerin (OPG) and death receptor 5 (DR5) with large-artery atherosclerosis (LAA) stroke and its prognosis.

Methods

We included patients with LAA stroke (n = 132) according to the TOAST classification system and controls (n = 60). To evaluate the extent and severity of cerebral atherosclerosis, the LAA stroke group was subdivided into 3 subgroups by number of cerebral arteries with atherosclerotic stenosis (≥50%): single, double and multiple (≥3). Plasma levels of TRAIL, OPG and DR5 were measured by ELISA. Ordinal logistic regression was used to analyze the association between the plasma levels of TRAIL, OPG, DR5 and the severity of cerebral atherosclerosis. Prognosis was determined by the Modified Rankin Scale at 3 months after stroke. Receiver operating characteristic (ROC) curve was used to evaluated TRAIL as a predictor of prognosis.

Results

Plasma TRAIL level was significantly lower for LAA patients than controls (P<0.001), while plasma OPG and DR5 levels were higher (both P<0.001). Logistic regression analysis revealed that risk of severe cerebral atherosclerosis was reduced significantly with increased plasma level of TRAIL (OR 0.438; 95% CI 0.282–0.681; P<0.001), whereas increased with high plasma levels of OPG and DR5 (OR 2.707; 95% CI 1.702–4.302, P <0.001; OR 3.593; 95% CI 1.878–6.869, P <0.001). Plasma TRAIL level was negatively correlated with the prognosis (r = - 0.372, P <0.001). The optimal cut-off value of TRAIL for prognosis was 848.63 pg/mL. The sensitivity and specificity at this cut-off value were 63.1% and 86.2%, respectively. After adding the plasma TRAIL level into the multivariate model of ROC, the area under the ROC curve was increased from 0.639 to 0.785, but the change was not statistical significant (P = 0.146).

Conclusions

TRAIL and its receptors OPG and DR5 may be involved in LAA stroke and the plasma level of TRAIL may be a biomarker predicting the severity of cerebral atherosclerosis and the prognosis of LAA stroke.     

The importance of abrogation of G2-phase arrest in combined effect of TRAIL and ionizing radiation

BACKGROUND: In this work we studied the relationship between the enhanced expression of DR5 receptor and the effect of combination of TRAIL and ionizing radiation on cell cycle arrest and apoptosis induction in human leukemia cell line HL-60. MATERIAL AND METHODS: DR5, APO2.7 and cell cycle were analyzed by flow cytometry. Proteins Bid and Mcl-1 were analyzed by Western-blotting. For clonogenic survival, colony assay on methylcellulose was used. RESULTS: Ionizing radiation caused significantly enhanced positivity of DR5 receptors 24 h after irradiation with high doses (6 and 8 Gy). An increase of DR5 receptor positivity after a dose of 2 Gy was not statistically significant and application of TRAIL 48 h after irradiation did not increase the apoptosis induction. However, a decrease of radiation-induced G(2) phase arrest and an increase of apoptosis were observed when TRAIL was applied 16 h before irradiation with the dose of 2 Gy. Incubation with 6 microg/l TRAIL for 16 h reduced D(0) value from 2.9 Gy to 1.5 Gy. The induction of apoptosis by TRAIL was accompanied by Bid cleavage and a decrease of antiapoptotic Mcl-1 16 h after incubation with TRAIL. CONCLUSION: TRAIL in concentration of 6 microg/l applied 16 h before irradiation by the dose of 1.5 Gy caused the death of 63% of clonogenic tumor cells, similarly as the dose of 2.9 Gy alone, which is in good correlation with the enhanced apoptosis induction.     

Specific resistance upon lentiviral TRAIL transfer by intracellular retention of TRAIL receptors

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in many transformed cells, suggesting TRAIL as an ideal candidate for cancer gene therapy. A main obstacle in cancer therapy is intrinsic or acquired therapy resistance of malignant cells. To study induction of resistance against TRAIL, we generated lentiviral vectors allowing efficient TRAIL expression and apoptosis induction in a variety of human cancer cell lines. Within days upon TRAIL overexpression, cells became resistant towards TRAIL, but not to CD95 ligation or DNA damage by cisplatin. Cell surface expression of TRAIL receptors 1 and 2 was completely abrogated in resistant cells due to intracellular retention of the receptors by TRAIL. SiRNA directed against TRAIL resensitized the resistant cells by restoring cell surface expression of TRAIL receptors. These findings represent a novel resistance mechanism towards TRAIL, specifically caused by TRAIL overexpression, and question the use of TRAIL expression in tumor-cell targeting gene therapy.     

Functional expression of TRAIL and TRAIL-R2 during human megakaryocytic development

The expression and function of surface TRAIL and TRAIL receptors were investigated in primary megakaryocytic cells, generated in serum-free liquid phase from peripheral human CD34(+) cells. The surface expression of both TRAIL and "death receptor" TRAIL-R2 became detectable starting from the early phase of megakaryocytic differentiation (day 6 of culture) and persisted at later (days10-14) culture times. On the other hand, "death receptor" TRAIL-R1, "decoy receptors" TRAIL-R3, and TRAIL-R4 were barely detectable or undetectable at any time point examined. Addition of recombinant TRAIL at day 6 of culture increased the rate of spontaneous apoptosis of CD34(+)/CD41(dim) megakaryoblasts and it significantly decreased the total output of mature megakaryocytic cells evaluated after additional 4-8 days of culture. Conversely, addition in culture of TRAIL-R2-Fc chimera, which blocked the interaction between endogenous TRAIL and TRAIL-R2 on the surface of cultured megakaryocytic cells, increased the total megakaryocytic cell count. In addition, recombinant TRAIL promoted a small but reproducible increase of maturation in the surviving megakaryocytic cell population, evaluated by both phenotypic analysis and morphology. A similar pro-maturation effect was observed when TRAIL was added to bone marrow-derived CD61(+) megakaryocytic cells. Thus, our data suggest a role of TRAIL as a regulator of megakaryocytopoiesis.     

Involvement of TRAIL/TRAIL-receptors in human intestinal cell differentiation

Despite the fact that tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and its receptors (TRAIL-Rs) are expressed in intestinal mucosa, little is known about the biological role of this system in intestinal cell physiology. The expression of surface TRAIL and TRAIL-R1, -R2, -R3, -R4 were examined by flow cytometry in the immortalized human cell line tsFHI under culture conditions promoting growth or growth arrest and expression of differentiated traits. A progressive increase of surface TRAIL expression paralleled tsFHI differentiation, consistently with immunohistochemistry analysis showing an increase of TRAIL immunostaining along the crypt-villus axis in normal jejuneal mucosa. In spite of the presence of TRAIL-R1 and TRAIL-R2 "death receptors," recombinant TRAIL was not cytotoxic for tsFHI cells. Exposure of tsFHI to recombinant TRAIL rather increased/anticipated the expression levels of the cyclin-dependent kinase inhibitors p21 and p27, which mediate the induction of growth arrest and the stabilization of differentiated traits, respectively, as well as of the canonical differentiation marker DPPIV. The differentiation inducing activity of TRAIL was abolished by pre-incubation with a Fc-TRAIL-R2 chimera. On the other hand, TRAIL did not significantly modulate the levels of osteoprotegerin (OPG), CXCL8/IL-8, CXCL9/MIG, and CXCL10/IP10 spontaneously released or induced by inflammatory cytokines. Taken together, these data suggest that TRAIL might act as a paracrine trophic cytokine on intestinal epithelium, promoting intestinal cell differentiation.