The Expressions And Interactions Of BCR Associated Factors And Their Signaling Pathways In B-CLL Cells

B cell Chronic lymphocytic leukaemia (B-CLL) is the commonest form of leukaemia, which develops in the ageing population. B-CLL is characterized by the progressive accumulation of functionally incompetent, mature looking, monoclonal CD5+, CD23+ B lymphocytes in blood, bone marrow, lymph nodes and spleen/liver. The etiopathogenesis of B-CLL in developmental immunology research remains unclear. But it has already found that the leukemic cells perform the evolution from a normal B cell to a clinically manifested B-CLL clone in vivo of B-CLL patient. And these processes influence by B cell receptror (BCR) and its signaling pathway which is critical in determining B cell fate.Recently, there are several advances have enhenced the understanding of BCR and BCR signaling pathway in B-CLL. Such as low level of surface BCR, mutational status of IGHV genes, and differential expression of ZAP70 and in differnet kinds of B-CLL. These advanves shed light on the abnormal regulation of BCR signaling pathway in B-CLL cells. Further studies to this project will offer clues for improving the design and targeting of therapeutic strategies.In this paper, we studied BCR related moleculars and signaling pathway. And we compared important differential expressions and interactions of several genes and proteins between B-CLL cells and normal peripheral B cells as well as in different B cells subgroup using western blotting, real-time RT-PCR, immunoprecipitation, flow cytometry and RNA interfere techniques, which will imporve the understanding of mechanisms for B-cell tumor pathogenesis and B-cell development.Expression of BCR inhibitory coreceptor CD22 related factors and signaling pathway via CD22 in B-CLL cells:Peripheral blood samples were collected from 63 typical B-CLL patients and 60 healthy donors. After increased the density of the unwanted cells by crosslinking antibody cocktail, we purified 83%±8% B cells by centrifugation over Ficoll buoyant density medium and isolated total protein and mRNA as research samples. We detected the protein expression of BCR inhibitory receptor CD22 by western blotting and N-Glycosidase F (PNGase F) which cleaves oligosaccharides of N-linked glycans on glycoproteins. Both CD22a and CD22βisoform are determined in CLL B cells, but the CD22 protein expression is less. We analyzed the phosphorylation of ITIMs motif in the cytoplasmic tails of CD22 in B-CLL cells compared with normal B cells, and found the significant decrease of active CD22 was dramatically. We determined that the recruitement of SHP-1 by CD22 was significant decreased using an co-immunoprecipitation approach, and further suggested BCR inhibitory pathway via CD22 is defective in B-CLL cells.Lyn proteins are overexpressed in B-CLL cells and block BCR funciton by phophrylated CD22. The results of real-time RT-PCR showed it does not depend on mRNA transcription. Both active Lyn and inactive Lyn are moderated up-regulation, but neither of them is as the same level as overexpressed total Lyn protein. Tyrosine kinase Syk is a key molecule in BCR active pathway, which requires phosphorylated by Lyn during activation. In B-CLL cells, Syk protein level is normal but activated phosphorylated Syk is increased, which means the constitutive activation of Syk downstream signaling pathway.Expression and function of c-Cbl E3 ubiquitin-ligase in B-CLL cells:In order to further understand the ubiquitination-proteasome system which targets BCR related proteins for degradation in B-CLL cells, we detected protein expressions of the system key factor c-Cbl E3 ubiquitin-ligase. The results of western blotting show the enhanced levels of c-Cbl protein, which is a post-transcriptional phenomena based on real-time RT-PCR results.We used an RNA interfere approach to transfect c-Cbl siRNA into in BJAB B cells, and detected surface CD22 expressions during gene knocking down process by flow cytometry. The results show that CD22 surface levels are upregulated. Enhanced c-Cbl expression in CLL B cells may therefor result in CD22 internalization and degradation. We co-immunoprecipated c-Cbl with phosphorylated tyrosine kinase, and determined a strong association between PI3-kinase P85 and c-Cbl in B-CLL This interaction is enhanced by increasing protein levels of B-CLL prognostic factor ZAP70, which suggest that p85 recuitment by c-Cbl may be an important regulation event in ZAP70 positive B-CLL. Differential expression of BCR related genes and proteins in peripheral blood B cells subgroups and B-CLL cells:To investigate the effect of BCR associated factors in B cell development and hypothesize the origins of CLL B cells, we separated naive B-cells, IgM memory B-cells (MZ B-cells) and switch memory B-cells by flow cytometry sorting using CD 19, CD27 and IgM as fluorescent marked phenotype. The purification rate is about 90%.B cell subgroups sorting was performed in 3 healthy donors and B-CLL patients samples, and 6 genes SIAE, c-Cbl, ST6Gall, ZAP70, Syk and Lyn were analysised by real-time RT-PCR using isolated mRNA as template. SIAE, c-Cbl is upregulated but ST6Gall is down-regulated in memory B cells. SIAE is an acetyl esterase that specifically removes acetyl moieties from 9-OH position of a2-6 liked sialic acid moieties to make ligands accessible to CD22. ST6GalI is a glycosyltransferase in the Golgi that can O-acetylated sialic acid-contaning ligands for CD22. The CD22 regulation by these two enzyme may be an characteristic of memory B cells. We also investigated protein levels of B-CLL overexpressed Lyn and c-Cbl in different kinds of B cells. The data indicates the up-regulation of Lyn protein may results from memory B cells accumulation following B-CLL patients aging. But c-Cbl protein overexperssion which is not regulated by gene may be an key event in B-CLL tumorigenesis.In conclusion, we constructed a hypothetical model about BCR associated signaling pathway based on research of BCR inhibitory and activation pathway and related proteins interaction in CLL B cells. In CLL B cells, following the low levels of surface BCR expression, CD22 is down-regulated but CD22 alpha and CD22 beta can be expressed equally well. At the same time, protein tyrosine Lyn is upregulated in CLL which included more active Lyn and inactive Lyn. More active Lyn contribute to phosphorylate protein tyrosine Syk that propagates the BCR signaling, while more inactive Lyn block phosphorylation of ITIMs in CD22, and results in abnormal recruit SHP-1, which results in defective BCR inhibitory signaling in B-CLL. E3 ligase c-Cbl is significant overexpressed in B-CLL, and affects the surface CD22 by leading it degradation. CD22 internalization in a c-Cbl dependent fashion becomes one of the most important phenotype of B-CLL. C-Cbl can phosphorylat regulatory subunit of PI3-kinase P85 which is associated with ZAP70 expression. This interaction may enhance activation of AKT and PKCδsignaling, and result in B-CLL cells abnormal accumulation. According to BCR related genes and proteins expression in B cell subgroups, SIAE and c-Cbl gene up-regulation and ST6Gall gene down-regulation could be potential molecular markers in memory B cells. However, Lyn protein levels enhanced may results from B-CLL patient aging, and c-Cbl protein overexpression may be an key event in tumorigenesis.