Notch2 is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia

Abstract

Members of theThe pathomechanisms underlying the disease primarily involve defects that prevent cell turnover because of apoptosis, rather than alterations in cell cycle regulation. 1,2 One of the hallmarks of B-CLL cells is the overexpression of the transmembrane glycoprotein CD23, 1-3 which undergoes spontaneous proteolysis, giving rise to soluble CD23 (sCD23) molecules. 4 The serum concentration of sCD23 can be several hundredfold higher than in healthy individuals and parallels the clinical stage of the disease. 5,6 Two isoforms of CD23 exist, CD23a and CD23b, which are expressed from 2 different promoters. 7 Expression of CD23a is restricted to B lymphocytes, whereas CD23b is found on a number of different hematopoietic cell types, predominantly after interleukin 4 (IL-4) treatment. 8 In B-CLL cells, selective expression of CD23a has been found to be concurrent to a state of cell survival, thereby providing a link between CD23a and the regulation of apoptosis. 9 CD23 is also closely associated with Epstein-Barr virus (EBV)-mediated B-cell immortalization, because a naturally occurring EBV mutant (P3HR1), carrying a deletion of the EBNA2 gene, has lost its ability to induce CD23 expression and to transform primary B cells in vitro. 10-12 EBNA2 activates the CD23a gene through a CBF1 repressor site located in the CD23a proximal promoter. [13][14][15] Several lines of evidence strongly suggest that EBNA2 mimics Notch signaling by acting as a transcriptional activator after binding and masking the repression domain of CBF1. [16][17][18][19] The Notch gene family encodes transmembrane receptors that modulate differentiation, proliferation, and apoptotic programs in response to extracellular ligands expressed on neighboring cells. 20,21 Ligand-mediated stimulation of Notch causes the proteolytic release of the intracellular domain (NotchIC), which then passes into the nucleus where it activates transcription of CBF1 responsive genes. [22][23][24][25] Deregulation of this pathway by overexpression of a constitutively activated form of Notch not only diverts cell fate decisions but is also tumorigenic. For example, truncation of Notch1 found in a subset of human T-cell leukemias leads to the expression of a ligand-independent oncogenic protein lacking the extracellular domain. 26 The truncated Notch proteins consist primarily of the intracellular domain and localize predominantly in the nucleus. Enforced expression of Notch1IC in bone marrow stem cells causes T-cell leukemia in mice, indicating a causative role for Notch1 in T-cell oncogenesis. 27 To elucidate the mechanisms leading to the up-regulation of CD23a in B-CLL cells, we analyzed the CD23a proximal promoter for sequence-specific DNA-protein interactions. By electrophoretic mobility shift assays (EMSAs), we detected a transcription factor For personal use only. on May 10, 2018. by guest www.bloodjournal.org From complex containing Notch2IC that binds to 5 different CBF1 responsive elements. Our data indicate that Notch2 is overexpressed in B-CLL cells, sug...

Induction of apoptosis by proteasome inhibitors in B-CLL cells is associated with downregulation of CD23 and inactivation of Notch2

“…An oligonucleotide derived from the CD23a promoter spanning a well-defined CBF-1-binding site was used as a probe. 36 In accordance with our previous results, 5 EMSA visualized a Notch2IC containing transcription factor complex (C1) in the nontreated control (Figure 3a and b). Incubation with PI MG132 (Figure 3a) or bortezomib (Figure 3b) resulted in a dosedependent reduction of the DNA bound Notch2IC complex.…”

“…Since CD23a is a target gene of Notch2 signaling and since nuclear extracts from freshly isolated B-CLL cells contain high amounts of activated Notch2IC, 5 we investigated whether the PI-mediated downregulation of CD23 is caused by a reduction in Notch2IC activity. EMSA were performed on nuclear extracts from B-CLL cells treated with PI (0.1-10 mM).…”

“…5 An oligonucleotide derived from the CD23a promoter (specific for the CD23a isoform) containing a CBF1-binding site (5 0 -CTCTAGTTCTCACCCAATTC-3 0 ) was annealed with the respective reverse complementary strand and used in the binding reactions. For analysis of NF-AT binding, an oligonucleotide with the sequence 5 0 -CGCCCAAAGAA-GAAAATTTGTTTCATA-3 0 was used.…”

“…4 We have recently shown that Notch2 is involved in the upregulation of CD23a expression in B-CLL cells. 5 Notch2 belongs to a family of short-range signaling transmembrane receptors, which participate in many binary cell fate decisions by modulating differentiation, proliferation, and apoptosis in response to extracellular stimuli. 6 After activation, the intracellular domain of Notch receptors (NotchIC) translocates to the nucleus where it activates CBF1 responsive genes.…”

Identification of Expression Signatures Predictive of Sensitivity to the Bcl-2 Family Member Inhibitor ABT-263 in Small Cell Lung Carcinoma and Leukemia/Lymphoma Cell Lines

“…FZD2 (frizzled homologue 2) is part of the Wnt pathway that includes the TCF7L1 gene that encodes the TCF-3 transcription factor, indicating that Wnt signaling may play a role in resistance. NOTCH2 is overexpressed in B-cell chronic lymphocytic leukemia and might also contribute to the inhibition of apoptosis in B-cell chronic lymphocytic leukemia (35), and constitutively active NOTCH2 decreases protease inhibitor-induced apoptosis in a human B-cell line (36).…”