hes1-protein--human and Multiple-Myeloma

The aim of this study was to explore the effect of DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycinet-butyl ester) on proliferation in vitro of human multiple myeloma cell line RPMI8226 and its underlying mechanism. The proliferation of RPMI8226 cells was detected by CCK-8 method; flow cytometry was employed to assay the cell apoptosis rate;the expressions of Notch1 and Hes1 proteins were detected by Western blot. The results indicated that the proliferation of human RPMI8226 cells significantly decreased after treatment with DAPT 0.5 - 5.0 µmol/L for 24 - 72 h (P < 0.05) in a concentration- and time-dependent manner. DAPT significantly induced apoptosis of RPMI8226 cells (P < 0.05). The expressions of Notch1 and Hes1 proteins were gradually downregulated with the increase of DAPT concentration. It is concluded that the DAPT can inhibit the proliferation of RPMI8226 cells, which may be related with the down-regulation of the protein expression of Notchl and Hes1.

Topics: Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Cell Line, Tumor; Cell Proliferation; Dipeptides; Homeodomain Proteins; Humans; Multiple Myeloma; Receptor, Notch1; Transcription Factor HES-1

Recent research has indicated that ligand-dependent activation of the Notch receptor in stromal cells plays a crucial role in the tumorigenesis of multiple myeloma. Ubiquitination of intracellular regions of Notch receptor and its ligands is important for Notch signal transduction. In vitro autoubiquitination analysis using recombinant proteins identified skeletrophin as a novel RING finger-dependent ubiquitin ligase. Skeletrophin bound the intracellular regions of the Notch ligand Jagged-2, but not to those of Delta-1, -3, -4, or Jagged-1. Skeletrophin, but not its RING-mutated form, ubiquitinized the intracellular region of Jagged-2. In malignant plasma cells from 23 of 40 multiple myeloma specimens, strong skeletrophin expression was observed, especially from patients with osteolytic bone lesions. Cytoplasmic localization, which may indicate Jagged-2 internalization, was found in many skeletrophin-positive myeloma cells. In contrast, skeletrophin was only weakly expressed in a few nonneoplasmic plasma cells in chronically inflamed tissues. Interestingly, exogenous expression of skeletrophin, but not the RING-mutated form, in Jagged-2-positive P3U1 myeloma cells induced Hes-1 (Hairy and Enhancer of Split homolog-1) gene expression in Notch receptor-positive bone marrow stromal cells through direct cell-cell contact. Thus, skeletrophin is a novel ubiquitin ligase that targets the intracellular region of Jagged-2 and is aberrantly overexpressed in multiple myeloma cells, possibly activating Hes-1 on stromal cells through ligand-dependent Notch signaling.

Topics: Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Bone Marrow Cells; Cells, Cultured; Coculture Techniques; Gene Expression; Homeodomain Proteins; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Jagged-2 Protein; Membrane Proteins; Microfilament Proteins; Models, Biological; Multiple Myeloma; Neoplasm Proteins; Polymerase Chain Reaction; Receptors, Notch; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Stromal Cells; Transcription Factor HES-1; Transfection; Ubiquitin-Protein Ligases