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.