| The Notch2, a member of the Notch family, is the major receptor for B-cell functions. Evidence demonstrates that the Notch2has various biological functions in lymphomas via its target genes, such as Nuclear factor kappa B (NF-κB). Very interestingly, there is complex cross-talk between Notch and NF-κB, and Notch2can affect the expression of the NF-κB, possibly as a feedback, the NF-κB signaling can also regulate the expression of the Notch2or components of the Notch2pathway. As the Notch2receptor is highly conserved, the mutations of Notch2were very rare in lymphomas. Until very recently, some Notch2mutations have been observed in some B-cell non-Hodgkin’s lymphoma (B-NHL) subtypes, such as Diffuse large B-cell lymphomas (DLCBLs) and marginal zone lymphomas (MZLs). DLCBL is the most common type of non-Hodgkin’s lymphomas, and it is well known to be highly heterogeneous both histologically and clinically. Today, the studies of DLBCLs are focusing on the better algorithm and therapeutic strategy of DLBCLs, which are based on the researches of the important oncogenes including Notch2. And this study focus is on the role of mutant Notch2in DLCBLs.First, we performed DNA sequencing of the exon26and the exon34of the Notch2gene in human DLBCLs. And we detected a identical mutation in the PEST (proline-, glutamic acid-, serine-and threonine-rich) domain of the Notch2gene in3of69 DLBCL cases. This mutation was mapped at the nucleotide7308(G/A) in the human Notch2cDNA sequence, which converted a tryptophan (TGG) into a termination codon (TGA), and led to partial deletion of the C-terminal PEST domain. Then we reported all three DLBCL cases with the Notch2mutation showed similar expression pattern, they were all prositive for Notch2, P50, P65and Ki67, which indicated the high rate of cell proliferation. Based on the mutation sequencing results above, the wild-type Notch2expression vector and mutant-type Notch2expression vector were constructed, and they then were transfected into the293T cell line and the lymphoma cell lines, including DLBCL cell lines (Pfeiffer, OCI-ly3and OCI-ly6) and Burkitt’s lymphoma cell lines (Raji, Ramos, Daudi and Namalwa). Next, the results of proliferation assay, cell-cycle assay and apoptotic assay showed that the mutant Notch2can trigger cell proliferation, accelerate the cell-cycle and resist apoptosis. Subsequently, the Co-Immunoprecipitation results showed that the mutant Notch2had higher binding affinity to the DNA-binding protein RBP-Jκ (recombination signal binding protein for immunoglobulin kappa J region, also called Su (H), CSL, or CBF1) than wild-type Notch2. And the transcriptional activity was significantly increased in mutant Notch2cells, compared with that in wild-type Notch2cells and controls. In addition, ectopic expression of the mutant Notch2receptor could activate NF-κB signaling, proved by Western Blot, Real-time PCR, and luciferase reporter. And through proliferation assay, we found that the proliferation induced by mutant Notch2was completely reversed by ammonium pyrrolidinedithiocarbamate (PDTC), an NF-κB signaling inhibitor. Finally, the Western Blot results showed that Notch2siRNA can regular the expression levels of NF-κB signal components, alternatively, PDTC also can affecte the expression level of Notch2.Altogether, these findings imply that DLBCL cases with Notch2mutations may stratify into a small subtype of diffuse large B-cell lymphoma, and they all exhibited strong cell proliferation signals. These data suggest that the PEST truncating Notch2enhances cell proliferation may through accelerating the cell-cycle and resisting apoptosis. And based on these genetic and phenotypic changes, we imply that the rapid proliferation induced by the truncate Notch2may be associated with the ectopic activation of both the Notch2signaling and the NF-κB signaling. Our results are expected to provide critical information for developing new DLBCL therapies by suppressing the Notch2and the NF-κB signaling. |
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