The truncate mutation of Notch2 enhances cell proliferation through activating the NF-κB signal pathway in the diffuse large B-cell lymphomas.

The Notch2 is a critical membrane receptor for B-cell functions, and also displays various biological roles in lymphoma pathogenesis. In this article, we reported that 3 of 69 (4.3%) diffuse large B-cell lymphomas (DLBCLs) exhibited a truncate NOTCH2 mutation at the nucleotide 7605 (G/A) in the cDNA sequence, which led to partial deletion of the C-terminal of PEST (proline-, glutamic acid-, serine- and threonine-rich) domain. The truncate Notch2 activated both the Notch2 and the NF-κB signals and promoted the proliferation of B-cell lymphoma cell lines, including DLBCL and Burkitt's lymphoma cell lines. Moreover, the ectopic proliferation was completely inhibited by ammonium pyrrolidinedithiocarbamate (PDTC), an NF-κB inhibitor. Simultaneously, PDTC also reduced the expression level of Notch2. Based on these results, we conclude that the Notch2 receptor with PEST domain truncation enhances cell proliferation which may be associated with the activation of the Notch2 and the NF-κB signaling. Our results are expected to provide a possible target for new DLBCL therapies by suppressing the Notch2 and the NF-κB signaling

The truncate Notch2 enhances cell proliferation which may be associated with the phosphorylation levels of the NF-κB signaling.

<p>(A) Western blotting and quantification of Notch2 (with c-myc tag), P65, p-P65, IκBα and p-IκBα were analyzed in the stably-infected Raji cells, OCI-ly3 cells and Pfeiffer cells (including wt Notch2 cells, mt Notch2 cells and pLVX cells). (B) MTT assay was used to detect the growth rates of the stably-infected Pfeiffer cells after treatment with 25 µM PDTC for 24 h, 48 h and 72 h. (C) Western blotting and quantification of Notch2 (with c-myc tag), P65, p-P65, IκBα, p-IκBα and β-actin in the stably-infected Pfeiffer cells were analyzed after treatment with 25 µM PDTC or the same dose of DMSO for 48 h. *<i>P</i><0.05 versus pLVX, #<i>P</i><0.05 versus wt Notch2, ▴<i>P</i><0.05 versus DMSO. Each experiment was performed independently for three times at least.</p

The truncate Notch2 enhances the cell proliferation.

<p>(A–G) MTT assay was used to detect the growth rates of the stably-infected lymphoma cell lines (Daudi, Raji, Ramos, Namalwa, OCI-ly3, OCI-ly6 and Pfeiffer), including wt Notch2 cells, mt Notch2 cells and pLVX cells. The MTT detected the absorbance (570 nm) at 24 h, 48 h, 72 h and 96 h. *<i>P</i><0.05 versus pLVX, while #<i>P</i><0.05 versus wt Notch2. Each experiment was performed independently for three times at least.</p

The immunohistochemical pattern of DLBCLs with the <i>NOTCH2</i> mutation.

<p>(A) The immunohistochemistry was used to analyze the immunohistochemical pattern of <i>NOTCH2</i> mutant DLBCLs with CD10, BCL6 and MUM-1 (×400 objective magnifications). (B) The immunohistochemistry was used to analyze the <i>NOTCH2</i> mutant DLBCLs (×400 objective magnifications) and the matched surrounding non-tumor tissues (×100 objective magnifications) with Notch2, P65, P50 and Ki67. But matched non-tumor tissue was not available in No. 445. (C) The positive rates of P50, P65, Ki67 and Notch2 were calculated in the two groups of DLBCLs (wt Notch2 and mt Notch2).</p

The mutant status of the <i>NOTCH2</i>.

<p>(A) The extracellular domain of Notch2 contains epidermal growth factor (EGF)-like repeats, three cysteine-rich LIN/Notch repeats (LNR) and heterodimerization domain (HD). The cytoplasmic domain contains RBP-Jκ associated molecule (RAM) domain, six ankyrin/CDC10 repeats (ANK) including two nuclear localization signals (NLS), Notch cytokine response (NCR) domain, transactivation domain (TAD) and PEST (proline-, glutamic acid-, serine- and threonine-rich) domain. There is a transmembrane domain (TM) between extracellular domain and cytoplasmic domain. The identical mutation of the three DLBCLs located at 2436^th amino acid in the PEST domain, which converted a tryptophan (TGG) into a termination codon (TGA), and resulted in a truncate Notch2. (B) The identical mutation of the three DLBCLs was at the nucleotide 7605 (G/A) in the human <i>NOTCH2</i> cDNA sequence. In the matched surrounding non-tumor tissues of both No. 505 and No. 646, the 7605 site showed “G” which was identical to the germline sequences; but matched non-tumor tissue was not available in No. 445.</p

The truncate Notch2 activates the NF-κB signaling.

<p>(A) The luciferase activity of the NF-κB signaling and (B) real-time PCR of P50 and P65 in lymphoma cell lines (Daudi, Raji, Ramos, Namalwa, OCI-ly3, OCI-ly6 and Pfeiffer) were analyzed after transfection or infection with the Notch2 (wt or mt) or the pLVX vectors. (C) Western blotting and quantification of Notch2 (with c-myc tag), P65, P50, IκBα and β-actin were analyzed in the stably-infected lymphoma cell lines including wt Notch2 cells, mt Notch2 cells and pLVX cells. *<i>P</i><0.05 versus pLVX, while #<i>P</i><0.05 versus wt Notch2. Each experiment was performed independently for three times at least.</p

The truncate Notch2 receptor increases the activity of the Notch2 signaling.

<p>(A) Co-IP was used to analyze the binding ability between Notch2 (with c-myc tag) and RBP-jκ (with HA tag). (B) The RBP-Jκ reporter and (C) the Hes1 reporter were used to detect the luciferase activity of the lymphoma cell lines (Daudi, Raji, Ramos, Namalwa, OCI-ly3, OCI-ly6 and Pfeiffer) after transfection with the Notch2 (wt or mt) vectors or the pLVX vectors. *<i>P</i><0.05 versus pLVX, while #<i>P</i><0.05 versus wt Notch2. Each experiment was performed independently for three times at least.</p