RESUMO
Notch signaling is often involved in cancer cell initiation and proliferation. Aberrant Notch activation underlies more than 50% of T-cell acute lymphoblastic leukemia (T-ALL); accordingly, chemicals disrupting Notch signaling are of potential to treat Notch-dependent cancer. Here, we developed a flow cytometry-based high-throughput assay to identify compounds that disrupt the interactions of DNA and RBPJ, the major downstream effector of Notch signaling. From 1492 compounds, we identified 18 compounds that disrupt RBPJ-DNA interactions in a dose-dependent manner. Cell-based assays further revealed that auranofin downregulates Notch-dependent transcription and decreases RBPJ-chromatin interactions in cells. Most strikingly, T-ALL cells that depend on Notch signaling for proliferation are more sensitive to auranofin treatment, supporting the notion that auranofin downregulates Notch signaling by disrupting RBPJ-DNA interaction. These results validate the feasibility of our assay scheme to screen for additional Notch inhibitors and provide a rationale to further test the use of auranofin in treating Notch-dependent cancer.
Assuntos
Auranofina/farmacologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Receptores Notch/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/efeitos dos fármacos , DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Ensaios de Triagem em Larga Escala/métodos , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptores Notch/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Epstein-Barr virus (EBV) is associated with a variety of human tumors. Although the EBV-infected normal B cells in vitro and the EBV-carrying B cell lymphomas in immunodeficient patients express the full set of latent proteins (type III latency), the majority of EBV-associated malignancies express the restricted type I (EBNA-1 only) or type II (EBNA-1 and LMPs) viral program. The mechanisms responsible for these different latent viral gene expression patterns are only partially known. IL-21 is a potent B cell activator and plasma cell differentiation-inducer cytokine produced by CD4(+) T cells. We studied its effect on EBV-carrying B cells. In type I Burkitt lymphoma (BL) cell lines and in the conditional lymphoblastoid cell line (LCL) ER/EB2-5, IL-21 potently activated STAT3 and induced the expression of LMP-1, but not EBNA-2. The IL-21-treated type I Jijoye M13 BL line ceased to proliferate, and this was paralleled by the induction of IRF4 and the down-regulation of BCL6 expression. In the type III LCLs and BL lines, IL-21 repressed the C-promoter-derived and LMP-2A mRNAs, whereas it up-regulated the expression of LMP-1 mRNAs. The IL-21-treated type III cells underwent plasma cell differentiation with the induction of Blimp-1, and high levels of Ig and Oct-2. IL-21 might be involved in the EBNA-2-independent expression of LMP-1 in EBV-carrying type II cells. In light of the fact that IL-21 is already in clinical trials for the treatment of multiple malignancies, the in vivo modulation of EBV gene expression by IL-21 might have therapeutic benefits for the EBV-carrying malignancies.
Assuntos
Linfócitos B/imunologia , Linfócitos B/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 4/genética , Interleucinas/farmacologia , Proteínas da Matriz Viral/genética , Linfócitos B/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Antígenos Nucleares do Vírus Epstein-Barr/farmacologia , Genoma Viral , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/efeitos dos fármacos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Linfoma de Células B/virologia , Neoplasias/genética , Neoplasias/virologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas da Matriz Viral/efeitos dos fármacos , Proteínas da Matriz Viral/farmacologia , Proteínas Virais/farmacologia , Latência Viral/genéticaRESUMO
UNLABELLED: Liver sinusoid (LS) endothelial cells (LSECs) support hepatocytes in resting livers and proliferate during liver regeneration to revascularize regenerated liver parenchyma. We report that recombination signal-binding protein-Jkappa (RBP-J), the critical transcription factor mediating Notch signaling, regulates both resting and regenerating LSECs. Conditional deletion of RBP-J resulted in LSEC proliferation and a veno-occlusive disease-like phenotype in the liver, as manifested by liver congestion, deposition of fibrin-like materials in LSs, edema in the space of Disse, and increased apoptosis of hepatocytes. Regeneration of liver was remarkably impaired, with reduced LSEC proliferation and destroyed sinusoidal structure. LSEC degeneration was obvious in the regenerating liver of RBP-J-deficient mice, with some LSECs losing cytoplasm, and organelles protruding into the remnant plasma-membrane of LSs to hamper the microcirculation and intensify veno-occlusive disease during liver regeneration. Hepatocytes were also degenerative, as shown by dilated endoplasmic reticulum, decreased proliferation, and increased apoptosis during liver regeneration. Molecular analyses revealed that the dynamic expression of several related molecules-such as vascular endothelial growth factor, vascular endothelial growth factor receptors 1 and 2, interleukin-6, and hepatocyte growth factor-was disturbed. CONCLUSION: Notch/RBP-J signaling may play dual roles in LSECs: in resting liver it represses proliferation, and in regenerating liver it supports proliferation and functional differentiation.
