A novel dysfunctional p53 mutation in the human neuroblastoma cell line TGW.

Mutations of p53 are rare in primary and advanced neuroblastomas. The p53 gene was studied in a TGW cell line established from a TNB1 xenograft, derived from metastasized neuroblastoma. The p53 protein level in TGW was elevated at baseline. Treatment with doxorubicin to induce genotoxic stress neither altered the p53 protein level nor induced p21 protein within 24 hours. DNA sequencing analysis revealed a novel triplet deletion mutation at codon 282 (R282del) of the p53 gene, a mutation also found in TNB1, indicating that the mutation occurred in the relapsed tumor. The mutant was incapable of transactivation and had no effect on the transactivational activity of the wild-type p53 gene product in reporter assays using a plasmid possessing a p53 responsive element of p21, bax or mdm2. These results suggest that the mutant p53R282del found in TGW is a non-functional mutant and has no dominant negative nature.

Multiple sites required for expression in 5'-flanking region of the hMLH1 gene.

Expression of the hMLH1 gene, one of the DNA mismatch repair genes, is frequently repressed in various cancers such as colorectal, ovarian, gastric, and endometrial origins with a microsatellite instable phenotype. In this study, we investigated details of the relationship between the transcriptional activity and the protein-binding sites in the 5'-flanking region of the hMLH1 gene. Luciferase reporter gene analysis with a series of deletion mutants revealed that a region containing -301 to -76 relative to a translation start site is essential for maximal expression. Eight protein-binding sites in this region were identified by in vivo methylation footprinting analysis and homology search. A presence of binding proteins to CCAAT-box at -145 to -139 was confirmed by the electrophoresis mobility shift assay. Partial involvement of NF-Y was seen by the super gel shift assay. Three reporter plasmids having a single site-directed mutation at -163 to -158, -145 to -139, or -96 to -93 showed 14-30% less activities to that of having the wild-type. Dual or triple mutations were no greater effect than the single mutation on the activity. These results indicate that three cis-elements are essential for full expression of the hMLH1 gene and may work co-operatively.

NATURE OF THE INHIBITION OF PROTEIN SYNTHESIS BY ETHIONINE AND AMINO ACID UPTAKE IN EARLY SEA URCHIN EMBRYOS.

The inhibition of protein synthesis by ethionine reported previously was found to be apparent, and ethionine inhibited only amino acid uptake like other usual amino acids. Even under such strong inhibition of the uptake, the syntheses of protein and DNA remained almost undiminished. The uptake of amino acid mixture by sea urchin embryos in the early cleavage stage was found to be carried out by active transport, since it was temperature-sensitive and was inhibited by 2,4-dinitrophenol. The uptake of an amino acid mixture or of single amino acids, e.g., valine, leucine and phenylalanine, was inhibited nonspecifically by an excess amount of other single amino acids added exogenously. Reflecting the inhibition of amino acid uptake, in vivo incorporation of amino acids into the protein fraction was apparently inhibited by excess amounts of other amino acids. As far as tested, the inhibition seems to be nonspecific and competitive for all amino acid species. The uptakes of leucine and phenylalanine were inhibited mutually by competition, with almost the same Km and Ki.