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Arsenic trioxide (As 2 O 3 ) induces clinical remission in acute promyelocytic leukemia, even in all-trans retinoic acidrefractory cases, with minimal toxicity at low (1^2 W WM) concentration. We exposed various neuroblastoma cell lines to As 2 O 3 at a concentration of 2 W WM: as a result, seven of 10 neuroblastoma cell lines underwent apoptosis characterized by morphological changes and nucleosomal DNA fragmentation. As 2 O 3 -induced apoptosis in neuroblastoma cells was shown to occur through the activation of caspase 3, as judged from Western blot analysis and apoptosis inhibition assay. It seemed that the sensitivity of neuroblastoma cells to As 2 O 3 was inversely proportional to their intracellular level of reduced glutathione. Taken together these results indicate that As 2 O 3 would be a candidate as a therapeutic agent for treatment of neuroblastoma, which is a solid tumor, not only by systemic therapy but also by local therapy.z 1999 Federation of European Biochemical Societies.

A cosmid library of the Escherichia coli K-12 W3110 chromosome was constructed in which clones were assigned to locations on the chromosome map by hybridization and genetic marker complementation tests. Approximately 70% of the genome was represented by this library. The identified clones can be maintained in the homologous system and would facilitate genetic studies of E. coli.Escherichia coli is one of the best-investigated microorganism in terms of gene structure and function, and more than 1,000 genes have been identified and localized on the circular chromosome map (2). Nevertheless, the number of genes characterized corresponds to only about one-third of the entire number estimated to be present on the E. coli chromosome. To understand the whole genetic system of E. coli, further isolation of genes with known genetic markers as well as a systematic search for new genes would be necessary. The first gene library of the E. coli chromosome was constructed by Clarke and Carbon in 1975 (10). Since then, the library has been screened to isolate clones which complement a variety of mutations at known locations on the genetic map. So far, about 300 clones have been mapped and analyzed by using this library (2, 36). In the work described in this paper, we attempted to construct an ordered cosmid library of the E. coli chromosome in which clones are assigned to their specific locations. The which appeared on ampicillin-containing agar plates were subjected to screening.For screening of clones, the -dot hybridization procedure involving isolated plasmid DNA was adopted to reduce the background level in hybridization. Although the colony

The N-myc amplification of human neuroblastomas was characterized by the amplified DNA cloned from the cell line MC-NB-1 using the phenol emulsion reassociation technique (PERT). A number of PERT clones exhibiting amplification in this cell line were tested for amplification in other neuroblastoma cell lines. In almost all cell lines examined, only a few clones were co-amplified with N-myc and most of the others were exclusively amplified in a subset of the cell lines. The total aggregate size of the Hind III fragment identified by the PERT clones was approximately 350 kb. Most of the PERT clones were mapped to human chromosome (chr) 2p23-2pter, where the N-myc gene is located. Four types of amplicons, the 100, 420, 480 and 520 kb fragments, shown to be Not I fragments, were identified by hexagonal field gel electrophoresis. Three fragments are ordered in a head-to-tail array, and the remaining fragment is either ordered in a tail-to-head array or something else. Despite the extremely unusual construction of the amplified sequences in this cell line as compared with others, there was a low degree of sequence heterogeneity among the amplicons within this cell line. These observations lead to the idea that the complex rearrangements that give rise to the heterogeneous organization of the amplified sequences among the different cell lines precede the amplification of these sequences.

A cDNA encoding a novel mucin protein, MUC20, was isolated as a gene that is up-regulated in the renal tissues of patients with immunoglobulin A nephropathy. We demonstrate here that the C terminus of MUC20 associates with the multifunctional docking site of Met without ligand activation, preventing Grb2 recruitment to Met and thus attenuating hepatocyte growth factor (HGF)-induced transient extracellular signal-regulated kinase-1 and -2 activation. Production of MUC20 reduced HGF-induced matrix metalloproteinase expression and proliferation, which require the Grb2-Ras pathway, whereas cell scattering, branching morphogenesis, and survival via the Gab1/phosphatidylinositol 3-kinase (PI3K) pathways was not affected. Thus, MUC20 reduces HGF-induced activation of the Grb2-Ras pathway but not the Gab1/PI3K pathways. We further demonstrate that the cytoplasmic domain of MUC20 has the ability to oligomerize and that the oligomerization augments its affinity for Met. Taken together, these results suggest that MUC20 is a novel regulator of the Met signaling cascade which has a role in suppression of the Grb2-Ras pathway.