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Newly emerging transformed cells are often eliminated from epithelial tissues. Recent studies have revealed that this cancer-preventive process involves the interaction with the surrounding normal epithelial cells; however, the molecular mechanisms underlying this phenomenon remain largely unknown. In this study, using mammalian cell culture and zebrafish embryo systems, we have elucidated the functional involvement of endocytosis in the elimination of RasV12-transformed cells. First, we show that Rab5, a crucial regulator of endocytosis, is accumulated in RasV12-transformed cells that are surrounded by normal epithelial cells, which is accompanied by upregulation of clathrin-dependent endocytosis. Addition of chlorpromazine or coexpression of a dominant-negative mutant of Rab5 suppresses apical extrusion of RasV12 cells from the epithelium. We also show in zebrafish embryos that Rab5 plays an important role in the elimination of transformed cells from the enveloping layer epithelium. In addition, Rab5-mediated endocytosis of E-cadherin is enhanced at the boundary between normal and RasV12 cells. Rab5 functions upstream of epithelial protein lost in neoplasm (EPLIN), which plays a positive role in apical extrusion of RasV12 cells by regulating protein kinase A. Furthermore, we have revealed that epithelial defense against cancer (EDAC) from normal epithelial cells substantially impacts on Rab5 accumulation in the neighboring transformed cells. This report demonstrates that Rab5-mediated endocytosis is a crucial regulator for the competitive interaction between normal and transformed epithelial cells in mammals.cell competition | endocytosis | apical extrusion | Rab5 | RasV12

Recent studies have revealed that newly emerging RasV12-transformed cells are often apically extruded from the epithelial layer. During this cancer preventive process, cytoskeletal proteins plectin and Epithelial Protein Lost In Neoplasm (EPLIN) are accumulated in RasV12 cells that are surrounded by normal cells, which positively regulate the apical elimination of transformed cells. However, the downstream regulators of the plectin-EPLIN complex remain to be identified. In this study, we have found that paxillin binds to EPLIN specifically in the mix culture of normal and RasV12-transformed cells. In addition, paxillin is accumulated in RasV12 cells surrounded by normal cells. Paxillin, plectin and EPLIN mutually influence their non-cell-autonomous accumulation, and paxillin plays a crucial role in apical extrusion of RasV12 cells. We also demonstrate that in RasV12 cells surrounded by normal cells, acetylated tubulin is accumulated. Furthermore, acetylation of tubulin is promoted by paxillin that suppresses the activity of histone deacetylase (HDAC) 6. Collectively, these results indicate that in concert with plectin and EPLIN, paxillin positively regulates apical extrusion of RasV12-transformed cells by promoting microtubule acetylation. This study shed light on the unexplored events occurring at the initial stage of carcinogenesis and would potentially lead to a novel type of cancer preventive medicine.

A series of mouse monoclonal antibodies (MAbs) to the nonstructural protein 3 (NS3) of hepatitis C virus was prepared. One of these MAbs, designated 8D4, was found to inhibit NS3 protease activity. This inhibition was competitive with respect to the substrate peptide (K i ‫؍‬ 39 nM) but was significantly decreased by the addition of the NS4A peptide, a coactivator of the NS3 protease. 8D4 also showed marked inhibition of the NS3-dependent cis processing of the NS3/4A polyprotein but had virtually no effect on the succeeding NS3/4A-dependent trans processing of the NS5A/5B polyprotein in vitro. Epitope mapping of 8D4 with a random peptide library revealed a consensus sequence, DxDLV, that matched residues 79 to 83 (DQDLV) of NS3, a region containing the catalytic residue Asp-81. Furthermore, synthetic peptides including this sequence were shown to block the ability of 8D4 to bind to NS3, indicating that 8D4 interacts with the catalytic region of NS3. The data showing decreased inhibition potency of 8D4 against the NS3/4A complex suggest that 8D4 recognizes the conformational state of the protease active site caused by the association of NS4A with the protease.Nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a multifunctional virus-specific protein that contains serine protease activity in its N-terminal region and accounts for processing of the viral polyprotein at four cleavage sites, NS3/ 4A, NS4A/4B, NS4B/5A, and NS5A/5B, whereas helicase and nucleic acid-stimulated nucleoside triphosphatase activities are found in its C-terminal region (see references 2 and 23 for reviews). The NS3 protease requires the NS4A protein as a cofactor for efficient cleavage of the polyprotein (35, 39). Because this enzyme plays an obligatory role in viral replication, it provides a logical target for the development of potentially selective antiviral agents. Development of increasingly specific inhibitors of NS3 requires detailed knowledge of the tertiary structure of the enzyme. X-ray crystallographic analysis (21,28,29,41) and nuclear magnetic resonance (NMR) spectroscopic analysis (1, 6) of the NS3 protease domain with or without the NS4A cofactor have provided a refined picture of the NS3 structure. Those studies show that the overall topology of NS3 protease is similar to that of chymotrypsin-like serine proteases and NS3 forms N-terminal (approximately residues 1 to 93) and C-terminal (residues 94 to 180) six-stranded antiparallel ␤-barrels that are packed like those of chymotrypsin-like serine proteases (1,6,21,28,29,41). The catalytic site of NS3 protease is formed by the triad of residues His-57, Asp-81, and Ser-139 and is found in the crevice between the two barrels. The interaction of NS4A with NS3 was shown to induce conformational changes in NS3 that involve both a structural reorganization of the N-terminal domain and a rearrangement of the protease catalytic site including 21,29). Although the tertiary structure of NS3 protease has been defined in detail, several loops found in other chymotrypsin family p...