A novel anti-human DR5 monoclonal antibody, TRA-8, induces apoptosis of most tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive tumor cells both in vitro and in vivo. In contrast to both the membrane-bound form of human TRAIL, which induced severe hepatitis in mice, and the soluble form of human TRAIL, which induced apoptosis of normal human hepatocytes in vitro, TRA-8 did not induce significant cell death of normal human hepatocytes. However, both primary hepatocellular carcinoma cells and an established liver cancer cell line were highly susceptible to the killing mediated by TRA-8. We show here that elevated levels of cell-surface expression of DR5 and increased susceptibility to DR5-mediated apoptosis are characteristics of malignant tumor cells. In contrast, DR5 alone is not sufficient to trigger apoptosis of normal hepatocytes. Therefore, selective, specific targeting of DR5 with an agonistic antibody might be a safe and effective strategy for cancer therapy.
The structure and hydrogen bonding of water in various kinds of aqueous polyelectrolyte solutions were analyzed with contours of O−H stretching of polarized Raman spectra. Effects of chemical properties of the polymers and water domains surrounded by the polymer chains on the relative intensity of collective band (C value) corresponding to a long-range coupling of O−H stretchings were discussed. The C values for various polymer solutions were almost constant in a relatively low molecular weight (M w) region, and decreased with an increase in M w value. When the size of the space surrounded by the pseudo-network was sufficiently small, the structure of water in the space was altered to have a relatively lower average number of hydrogen bonds between water molecules than that of bulk water. The number of hydrogen bonds collapsed by the presence of one monomer residue (N value) of polyelectrolyte (sodium polyethylenesulfonate, poly-l-lysine hydrobromide, etc.) with a small M w was much larger than those for neutral polymers such as poly(ethylene glycol) and poly(N-vinylpyrrolidone). This result indicates that the monomer residues of water-soluble neutral polymers do not disturb the structure of water significantly, whereas electrostriction effect by the polyelectrolyte is quite effective on the structure of water. On the contrary, the N value for poly(2-methacryloyloxyethyl phosphorylcholine) with a small M w was nearly zero, suggesting that the zwitterionic-type monomer residues do not disturb the hydrogen bonding between water molecules.
Recent evidence indicates that the transactivation of estrogen receptor a (ERa) requires estrogen-dependent receptor ubiquitination and degradation. Here we show that estrogen-unbound (unliganded) ERa is also ubiquitinated and degraded through a ubiquitin-proteasome pathway. To investigate this ubiquitin-proteasome pathway, we purified the ubiquitin ligase complex for unliganded ERa and identified a protein complex containing the carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP preferentially bound to misfolded ERa and ubiquitinated it to induce degradation. Ligand binding to the receptor induced the dissociation of CHIP from ERa. In CHIPÀ/À cells, the degradation of unliganded ERa was abrogated; however, estrogen-induced degradation was observed to the same extent as in CHIP þ / þ cells. Our findings suggest that ERa is regulated by two independent ubiquitin-proteasome pathways, which are switched by ligand binding to ERa. One pathway is necessary for the transactivation of the receptor and the other is involved in the quality control of the receptor.
The 2-5A system is one of the major pathways for antiviral and antitumor functions that can be induced by interferons (IFNs). The 2-5A system is modulated by 5-triphosphorylated, 2,5-phosphodiester-linked oligoadenylates (2-5A), which are synthesized by 2,5-oligoadenylate synthetases (2,5-OASs), inactivated by 5-phosphatase and completely degraded by 2-phosphodiesterase (2-PDE). Generated 2-5A activates 2-5A-dependent endoribonuclease, RNase L, which induces RNA degradation in cells and finally apoptosis. Although 2,5-OASs and RNase L have been molecularly cloned and studied well, the identification of 2-PDE has remained elusive. Here, we describe the first identification of 2-PDE, the third key enzyme of the 2-5A system. We found a putative 2-PDE band on SDS-PAGE by successive six-step chromatographies from ammonium sulfate precipitates of bovine liver and identified a partial amino acid sequence of the human 2-PDE by mass spectrometry. Based on the full-length sequence of the human 2-PDE obtained by in silico expressed sequence tag assembly, the gene was cloned by reverse transcription-PCR. The recombinant human 2-PDE expressed in mammalian cells certainly cleaved the 2,5-phosphodiester bond of 2-5A trimer and 2-5A analogs. Because no sequences with high homology to this human 2-PDE were found, the human 2-PDE was considered to be a unique enzyme without isoform. Suppression of 2-PDE by a small interfering RNA and a 2-PDE inhibitor resulted in significant reduction of viral replication, whereas overexpression of 2-PDE protected cells from IFN-induced antiproliferative activity. These observations identify 2-PDE as a key regulator of the 2-5A system and as a potential novel target for antiviral and antitumor treatments.
CS-1008 has a selective toxicity toward tumor cells expressing DR5 and the potential for antitumor efficacy in human malignancies.
TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.
The state of sorbed water and sorption processes of water in poly(ethylene glycol) (PEG) films were studied by Fourier transform infrared. From the assignment and time evolution of the components of the O-H stretching band, the adsorption and penetration of water into the films are considered to proceed as follows: (1) At the polymer surface, a water molecule binds to the oxygen atoms of PEG with one of its hydrogen atoms ("binding water"). The same phenomenon more gradually occurs in the polymer matrix too. (2) The water binding to the oxygen atoms of PEG molecules with both of its hydrogen atoms ("bridging water") is formed from the binding water, while a dimeric water is gradually formed by the association of the free water molecule with the binding water. (3) The amount of monomeric species (both binding water and bridging water) is equilibrated. (4) The water dimer is further formed by the association of the free water molecule with the binding water, and finally the amount of the dimer is equilibrated. When the molecular weight of PEG was small, a new band at 3400 cm -1 gradually appeared. This band could be attributed to the O-H stretching of water molecules attaching to hydroxyl groups at the end of PEG molecules. An ab initio molecular orbital calculation and hybrid density functional method supported the assignment of the peaks. Experimental SectionMaterials. PEGs [viscosity-averaged molecular weight (Mv) ) 1.1 × 10 4 (11K), 1.5 × 10 4 (15K), 2.8 × 10 4 (28K), 4.9 × 10 4 (49K), and 6 × 10 4 (60K)] and PEG-DME [Mw ) 2.1 × 10 3 (2.1K)] from Aldrich, Milwaukee, WI, were purified by precipitation in ethanol-diethyl ether. Poly(methyl methacrylate) (PMMA; atactic, Mv ) 1.23 × 10 5 ) from Wako Pure Chemicals, Osaka, Japan, was used without further purification. Poly(2-hydroxyethyl methacrylate) (PHEMA; Mv ) 6.4 × 10 4 ) was prepared by the conventional radical polymerization of 2-hydroxyethyl methacrylate (HEMA; purified by distillation in vacuo) in methanol at 65 °C for 24 h using 2,2′-azobis(isobutyronitrile) (AIBN) as the initiator (molar ratio of HEMA and AIBN, 100:1) and subsequent precipitation from diethyl ether. Polyethylene film (PE; low-density polyethylene; thickness, 10 µm) from Ohji Special Carton Co., Tokyo, Japan, was used without further purification. The Mv values of PEG, PMMA, and PHEMA were determined by using an Ubbelohde dilution-type viscometer (type 0B; Kusano,
The structure of water sorbed into poly(2-methoxyethyl acrylate) (PMEA), poly(2-hydroxyethyl methacrylate) (PHEMA), and their copolymers (p(MEA/HEMA)) was investigated by attenuated total reflection infrared (ATR-IR) spectroscopy. The extinction coefficient of the OH stretching band of sorbed water ( OH) was calculated from the band area obtained by IR measurement and the amount of sorbed water obtained by thermogravimetric analysis. When the polymers contacted with water vapor (relative humidity ) ∼55%), the OH values were quite similar in all polymers. On the other hand, when the polymers contacted with liquid water, the OH values were drastically changed by the content of 2-methoxyethyl acrylate (MEA). When the MEA content of the polymers was low (<60 mol %), the OH value of the water sorbed into polymers in contact with liquid water was equal to that in contact with water vapor. In the higher MEA content (70-100 mol %), on the other hand, the OH values of the water sorbed into polymers in contact with liquid water were 5-8 times larger than that in contact with water vapor. These results seemed to indicate that the interaction between the primary hydration water around the MEA-containing polymer chain and water molecules surrounding the primarily hydrating water is very weak. Such water with a large OH value seemed to correspond to "cold-crystallizable" water, which has been observed by DSC as anomalous water other than intermediate and nonfreezable waters. Taking both the experimental results obtained in this work and those thermodynamically obtained previously into consideration, it was strongly suggested that the cold crystallization of water is generated by caging water molecules in a small space by the polymer chains with a small hydration region. The correlation between the OH value and the blood compatibility of the copolymer was also discussed.
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