Notch and BCR signaling synergistically promote the proliferation of Raji B-lymphoma cells

Abstract

The evolutionarily conserved Notch signaling pathway plays a pivotal role in cell proliferation, apoptosis, and cell fate decision from invertebrates to vertebrates, and is oncogenic in some human hematopoietic malignancies. To study the role of Notch signaling in B-lymphoma, we expressed a soluble fragment of human Delta-like1 (hDll1) in E. coli, which was shown to activate the Notch signaling. Incubation of Burkitt’s lymphoma Raji cells with the soluble hDll1 led to γ-secretase-dependent up-regulation of a Notch downstream gene, Hes1. This treatment synergized with B-cell receptor (BCR)-mediated signaling to promote proliferation of Raji cells in vitro, which was cancelled by GSI. We further showed that Notch signaling significantly repressed, while γ-secretase inhibitor (GSI) enhanced, “natural” apoptosis of Raji cells. Because c-myc is a downstream gene of both Notch signaling and BCR signaling, and GSI blocked c-myc expression in the presence of hDll1 and anti-IgM, Notch signaling might interact with BCR signaling at the level of c-myc expression to regulate proliferation and apoptosis of B-lymphoma cells.

Introduction

The Notch signaling pathway has multiple and essential roles in cell fate decisions in development and is conserved during evolution from worm through man [1]. The Notch pathway in mammals includes four receptors, Notch1-4, and five ligands including Jagged1, 2 and Delta-like1, 3, 4. When Notch receptor is triggered by direct interaction with its ligands, expressed on neighboring cells, the intracellular domain of the Notch receptor (NIC) is released from the membrane after sequential receptor cleavages executed by ADAM/TACE protease and γ-secretase complex [2], [3]. NIC translocates into nucleus and associates with the transcription factor RBP-J, and transactivates promoters harboring RBP-J-binding sites through recruiting co-activators of the Mastermind-like (MAML) family [4], [5]. The extracellular domains, including an N-terminal Delta-Serrate-Lag2 (DSL) domain that is essential for Notch-binding, and varying number of EGF-like repeats, of Notch ligands are evolutionarily conserved. The human Delta-like1 (hDll1) is a homolog of the Drosophila Delta, and is composed of 723 amino acids, with one DSL domain and eight EGF-like repeats [1].

During mammalian hematopoiesis and lymphopoiesis, Notch signaling shows multiple functions, such as the regulation of T-cell commitment from a common lymphoid precursor at the expense of B-cell development in thymus [6], and the control of marginal zone B-cell differentiation in spleen [7]. Notch signaling must be precisely regulated, as constitutive Notch signaling leads to T-cell malignancies [8]. In the B-cell compartment, the effect of de-regulating Notch signaling in the B-cell lineage is more complicated and controversial [9]. Although there has been no report showing that constitutive activation of Notch signaling lead to B-cell malignancies, activation of the transcription factor RBP-J by EBNA2 is essential for B-cell transformation by Epstein-Barr (EB) virus [10], [11].

B-cell antigen receptor (BCR) plays an important role in the regulation of proliferation, apoptosis, and differentiation of B-cells at distinct stages during B-cell development. It has been shown that Notch signaling synergizes with BCR to enhance B-cell activation [12]. But, it is not clear how BCR and Notch signaling interacts in B-cell malignancies. In this report, using a human Burkitt’s lymphoma, the Raji cells, we showed that BCR-mediated proliferation was enhanced by co-stimulating Notch receptors with a recombinant hDll1 protein, and that BCR and Notch signaling might interact at the level of c-myc expression.