n-(n-(3-5-difluorophenacetyl)alanyl)phenylglycine-tert-butyl-ester and Lymphoma--B-Cell

Notch-signalling has been implicated as a pathogenetic factor and a therapeutical target in T-cell leukaemias and in some lymphomas of B-cell origin. Our aim was to investigate the role of Notch-signalling in apoptosis regulation in human non-Hodgkin B-cell lymphoma (B-NHL) cell lines and in primary chronic lymhocytic leukaemia (CLL) cells using Delta-like 4 (Dll4) ligand mediated Notch activation and gamma-secretase inhibitor (GSI) mediated Notch inhibition in vitro. The potential cross-talk of Notch with the transforming growth factor-beta (TGFb) pathway in apoptosis induction was also explored, and the effect of GSI on drug-induced apoptosis was assessed. Modulation of Notch-signalling by itself did not change the rate of apoptosis in B-NHL cell lines and in CLL cells. TGFb-induced apoptosis was decreased - but not completely abolished - by GSI in TGFb-sensitive cell lines, but resistance to the apoptotic effects of TGFb were not reversed by Notch activation or inhibition. Drug-induced apoptosis was not modified by GSI. We identified Hairy/Enhancer of Split (HES)-1 as a TGFb target gene in selected - TGFb-sensitive - B-NHL cell lines. TGFb-induced HES-1 was only partially Notch-dependent in later phases. Apoptosis regulation by TGFb and GSI was not dependent on the transcriptional regulation of c-myc. In conclusion, our data does not support a unifying role of Notch in regulating apoptosis in B-NHL, but warns that gamma-secretase inhibitors may actually counteract apoptosis in some cases.

Topics: Adaptor Proteins, Signal Transducing; Amyloid Precursor Protein Secretases; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Calcium-Binding Proteins; Cell Line, Tumor; Dipeptides; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Receptors, Notch; Repressor Proteins; Signal Transduction; Transcription Factor HES-1; Transforming Growth Factor beta

Ectopic Myc expression plays a key role in human tumorigenesis, and Myc dose-dependent tumorigenesis has been well established in transgenic mice, but the Myc target genes that are dependent on Myc levels have not been well characterized. In this regard, we used the human P493-6 B cells, which have a preneoplastic state dependent on the Epstein-Barr viral EBNA2 protein and a neoplastic state with ectopic inducible Myc, to identify putative ectopic Myc target genes. Among the ectopic targets, JAG2 that encodes a Notch receptor ligand Jagged2, was directly induced by Myc. Inhibition of Notch signaling through RNAi targeting JAG2 or the gamma-secretase Notch inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT) preferentially inhibited the neoplastic state in vitro. Furthermore, P493-6 tumorigenesis was inhibited by DAPT in vivo. Ectopic expression of JAG2 did not enhance aerobic cell proliferation, but increased proliferation of hypoxic cells in vitro and significantly increased in vivo tumorigenesis. Furthermore, the expression of Jagged2 in P493-6 tumors often overlapped with regions of hypoxia. These observations suggest that Notch signaling downstream of Myc enables cells to adapt in the tumor hypoxic microenvironment.

Topics: Animals; B-Lymphocytes; Cell Hypoxia; Cell Proliferation; Cell Transformation, Neoplastic; Dipeptides; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Humans; Intercellular Signaling Peptides and Proteins; Jagged-2 Protein; Lymphoma, B-Cell; Membrane Proteins; Mice; Models, Biological; Proto-Oncogene Proteins c-myc; Receptors, Notch; RNA Interference; Transcriptional Activation

A novel lymphoma cell line, designated TMD8 was established from cells of a patient with diffuse large B-cell lymphoma. TMD8 cells expressed HES1 mRNA, suggesting constitutive activation of Notch signaling. TMD8 cells expressed normal-sized Notch1 protein, and showed no mutations in the NOTCH1 gene. Cell growth was suppressed by gamma-secretase inhibitors (GSI). It was reported that GSI suppressed growth of T-cell acute lymphoblastic leukemia (T-ALL) cell lines, which frequently had NOTCH1 mutations. In addition to T-ALL, TMD8 is another unique cell line sensitive to GSI, and is useful to study effects of GSI in molecular targeting therapy.

Topics: Adaptor Proteins, Signal Transducing; Amyloid Precursor Protein Secretases; Basic Helix-Loop-Helix Transcription Factors; Calcium-Binding Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dipeptides; Enzyme Inhibitors; Fatal Outcome; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Jagged-2 Protein; Karyotyping; Ligands; Lymphoma, B-Cell; Male; Membrane Proteins; Middle Aged; Oligopeptides; Receptor, Notch1; Receptor, Notch2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Structure-Activity Relationship; Transcription Factor HES-1