Aberrant Regulation of the LEF-1 Locus in Monoclonal B Cell Lymphocytosis (MBL) and Chronic Lymphocytic Leukemia (CLL): A Possible Role for Epigenetic Regulation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 669-669
Author(s):  
Albert Gutierrez ◽  
Renee Tschumper ◽  
Tait D. Shanafelt ◽  
Jeanette Eckel-Passow ◽  
Neil E. Kay ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cells ◽  
B Cell ◽  
Promoter Region ◽  
Cpg Island ◽  
Lymphocytic Leukemia ◽  
Human B Cells ◽  
Genome Wide ◽  
Normal Human ◽  
Expression Status

Abstract Abstract 669 Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world but its pathogenesis remains largely unknown. More recently, a widely prevalent premalignant condition termed monoclonal B cell lymphocytosis (MBL) has been defined and similarly involves expansion of a CD19+CD5+ population of B cells. MBL has been the focus of a wide number of recent studies in hopes it can provide insight into the early pathological events that lead to clonal expansion of a pre-leukemic CLL-like clone. Previously, we and others identified the transcription factor lymphocyte enhancer-binding factor-1 (LEF-1) as one of several genes significantly over expressed in CLL B cells relative to blood CD19+ B cells from healthy adults. LEF-1 is crucial for the proliferation and survival of pro-B cells during B cell development and deregulated LEF-1 activation has been directly linked with leukemogenesis in a transgenic murine model. In the present study, we addressed three critical questions concerning LEF-1 and CLL: 1) do CD5+ normal B cells express LEF-1; 2) at what stage of transformation does LEF-1 expression first appear; and 3) what mechanism(s) underlies LEF-1 expression in leukemic B cells. Methodology: The goals of our study were to: 1) determine the LEF-1 expression status of normal CD 19+/CD5+ human B cells; 2) determine the expression status of LEF-1 in clonal B cells present in patients with MBL; and 3) identify regulatory mechanisms that control aberrant expression of LEF-1 in CLL B cells. In order to achieve the first two goals, a 3-color flow cytometry assay for CD19, CD5, and intracellular LEF-1 was developed. To achieve the third goal, we performed in silico analysis of the LEF-1 locus and correlated this with publicly available genome wide methylated CpG island recovery assay (MIRA) data describing the methylome of normal human B cells (Rauch, T. A. Proc Natl Acad Sci U S A. 2009; 106(3): 671-8). Results: Analysis of human umbilical cord blood B cells, a rich source of the CD19+/CD5+ B cell subset, revealed that all normal CD 19+/CD5+ human B cells are negative for LEF-1 protein expression. These data demonstrate LEF-1 expression by CLL B cells is truly aberrant and does not simply reflect the phenotype of CD5+ lineage B cells. We next tested if the CD19+/CD5+ cells obtained from patients with MBL express LEF-1 protein. In these analyses, we restricted our study to MBL patients with absolute B cell counts of less than 2.5 ×10 9 cells/(L) (range: 0.756 - 2.44 × 10 9 cells/(L)), which is significantly below the upper limit that defines the MBL to CLL transition. Of interest, each MBL sample analyzed (n=6) revealed the presence of two populations of cells: 1) CD19+/CD5+ B cells expressing LEF-1; and 2) CD19+/CD5- B cells lacking expression of LEF-1. To confirm the clonal nature of the CD19+/CD5+ cells from MBL patients we demonstrated that the cells were light chain restricted. These data clearly indicated that LEF-1 expression becomes deregulated in the premalignant state of MBL and may therefore represent an early event in CLL leukemogenesis. In order to identify differential pathways of LEF-1 regulation that may be altered in CLL and MBL B cells, in silico promoter analysis of LEF-1 was performed. We identified a number of potential transcription factor binding sites as well as a putative CpG island in the 5' promoter region of LEF-1. Using data from a human B cell genome wide methylation array, we were able to determine that this same putative LEF-1 promoter CpG island was highly methylated in normal human B cells. Methylation of promoter region CpG islands is known to play an important role in developmental regulation of gene expression and may be the operative mechanism underlying silencing of LEF-1 expression in normal B cells. Conclusions: LEF-1 expression is deregulated in MBL and appears to be an early and possibly key event in the transition of normal B cells into a premalignant/malignant state. Our data suggest that loss of epigenetic regulation of this developmentally important locus may play a role in aberrant LEF-1 expression in MBL and CLL B cells. Ongoing studies are aimed at determining the methylation status of the LEF-1 promoter in CLL B cells and the functional role of this protein in transcriptional regulation and survival of CLL and MBL cells. Disclosures: No relevant conflicts of interest to declare.

Upregulation of Human Pim-2 in B-CLL, Involves Interactions between Oct-1, Oct-2, Bob-1 and NF-kB.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1916-1916
Author(s):  
Yosef Dicken ◽  
Amos M. Cohen ◽  
Hanna Bessler ◽  
Daphna Levi-Hirsh ◽  
Ariela Arad ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cells ◽  
B Cell ◽  
Promoter Region ◽  
Lymphocytic Leukemia ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Cell Extracts ◽  
Hodgkin's Lymphomas

Abstract hPim-2 is a proto-oncogene that encodes a serine/threonine kinase and inhibits apoptosis by phosphorylation of BAD. We have shown that hPim is upregulated in human non-Hodgkin’s lymphomas (NHL) and in chronic lymphocytic leukemia (B-CLL) and its cellular transcript levels in B-CLL correlates with lymphocyte doubling time. We found no mutations in the promoter region of hPim-2 in B-cells of 30 patients with CLL (~2000 bp upstream). The proximal promoter region of hPim-2 (600 bp) contains two adjacent NF-kB-binding elements, two adjacent Oct-binding elements and an SP1 element by bioinformatic analysis. Studies have recently shown that the transcription factor Oct-2 and the B-cell specific Oct cofactor Bob-1 are overexpressed in certain large B-cell lymphomas, whereas increased expression of Bob-1 has also been observed in T-cell neoplasms. Shift assays (EMSA) analysis, using nuclear extracts from B-CLL cells and various fragments of hPim-2 promoter region used as probes, revealed that complexes containing an Oct elements were consistently heavier in B-CLL extracts compared with control B-cells. Accordingly, Oct-1, Oct-2 and Bob-1 protein levels were significantly higher in B-CLL compared to healthy extracts. Moreover, chromatin immunoprecipitation (Chip) assays confirmed that in-vivo Oct-1+2 and Bob-1 are indeed physically attached to the hPim-2 promoter, and that this interaction is significantly more intensive in B-CLL cells than in control B-cells. Furthermore, we have found in addition that the p52 isoform subunit of NF-kB predominates the interaction with the kB element in the hPim-2 promoter in B-CLL cells, as compared to the p50 isoform observed in control B-cells. To determine whether these interactions are transcriptionaly significant, we fused the luciferase reporter gene to various promoter fragments, and monitored luciferase expression in-vitro after incubation with either B-CLL or normal B-cell extracts. Luciferase expression was consistently higher when Oct element-containing fragment was incubated with B-CLL cell extracts. Together, these results suggest that the upregulation of hPim-2 in B-CLL is due to enhanced expression and transcriptional activity of the Oct-1+2 and Bob-1 complex and that it might synergistically act with the p52 containing NF-kB transcription factor.

ZAP-70 Expression Associated with Activation in Normal Human B Cells and B Cell Chronic Lymphocytic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2794-2794 ◽  
Author(s):  
Renee C. Tschumper ◽  
Jeffrey C. Nolz ◽  
Neil E. Kay ◽  
Cheryl A. Jankiewicz ◽  
Diane F. Jelinek
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Type B ◽  
Human B Cells ◽  
Normal Human ◽  
Cell Chronic Lymphocytic Leukemia

Abstract The protein tyrosine kinase ZAP-70, known to be critical for T cell development and T cell receptor signaling, has become a potential prognostic tool for B cell chronic lymphocytic leukemia (B-CLL). Recent studies have suggested that those patients with ZAP-70 positive leukemic cells have a poorer disease prognosis than do patients whose leukemic cells lack ZAP-70 expression. In addition, ZAP-70 expression occurs more frequently in CLL B cells that express unmutated immunoglobulin (Ig) VH region genes. However, several questions remain regarding ZAP-70 expression in B-CLL. First, is ZAP-70 expression in some patients with B-CLL the consequence of malignant transformation or do normal human B cells express ZAP-70 under certain conditions of stimulation? Secondly, does the expression of ZAP-70 reflect a more activated phenotype, thus resulting in the more aggressive phenotype of ZAP-70+ B-CLL? To begin to address these questions, we focused our studies on ZAP-70 expression in normal, human B cells. CD19+ B cells were isolated by magnetic bead separation from normal human peripheral blood (PB) or tonsil and spleen surgical waste tissue. We first used three-color flow cytometry to evaluate ZAP-70 expression in CD19+ versus CD3+ lymphocytes. While PB B cells were negative for ZAP-70 expression, we were able to detect a subset of CD19+/ZAP-70+ cells in both spleen and tonsil and this subset expressed ZAP-70 at levels comparable to those of CD3+ T cells. To verify the flow cytometry results, we evaluated total ZAP-70 expression by immunoblotting and observed readily detectable ZAP-70 in both splenic and tonsillar CD19+ cells. Based on the observation that ZAP-70 was found in B cells of tonsil and spleen, but not PB, we hypothesized that ZAP-70 may be associated with activation status since tonsillar and splenic B cells will include a population of activated B cells. To examine this issue, we sorted CD19+/CD38+ from CD19+/CD38− tonsillar cells and again evaluated ZAP-70 expression. Whereas both populations expressed ZAP-70, CD38+ B cells expressed ZAP-70 at a higher level suggesting that ZAP-70 may be associated with a more activated phenotype. To address the issue of activation, we activated CD19+ PB B cells with a cocktail of CD40L, IL-10, IL-4 and IL-6 for 3 days and analyzed ZAP-70 expression. Indeed, we were able to induce ZAP-70 expression in a subpopulation of blood B cells. We also assessed ZAP-70 phosphorylation following tonsillar B cell receptor (BCR) ligation and we observed that phosphorylation of ZAP-70 (tyrosine 493) occurred within 10 min. of BCR stimulation. Finally, we assessed whether ZAP-70 expression in B-CLL could be associated with an activated cell phenotype. By stimulating Ig VH non-mutated and mutated B-CLL patient samples with the CD40L cocktail, we observed an increase in ZAP-70 expression in non-mutated CLL by immunoblotting. These results demonstrate expression of ZAP-70 in a subset of normal B cells that express an activated phenotype. Moreover, our results also suggest that ZAP-70 levels in CLL B cells may also be increased following cellular activation. Given that non-mutated type B-CLL expresses ZAP-70 more frequently than mutated type B-CLL, ZAP-70 expression may simply reflect a more activated population of leukemic cells that may correlate with progressive disease. Continuing investigation into the role of ZAP-70 in normal B cell development and B-CLL is clearly warranted.

ZAP-70 Expression in Human B cells: Analysis of Normal Cells and Acute Lymphoblastic Leukemia (ALL) Cells with Pro/Pre B Phenotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4443-4443
Author(s):  
Marta Crespo ◽  
Neus Villamor ◽  
Eva Gine ◽  
Dolors Colomer ◽  
Teresa Marafioti ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Protein Tyrosine Kinase ◽  
Lymphoblastic Leukemia ◽  
Human Lymphocytes ◽  
Critical Role ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Qrt Pcr ◽  
Human B Cells

Abstract ZAP-70 is a protein tyrosine kinase of the Syk/ZAP-70 family that plays a critical role in the signal transduction from the T-cell receptor. In human lymphocytes, ZAP-70 gene has been reported to be expressed in T and NK derived cells, and in IgVH unmutated B-chronic lymphocytic leukemia cells. More recently, ZAP-70 expression has been shown to be required for the development of pro-B cells to pre-B cells in mice. To ascertain the expression of ZAP-70 gene in human immature B-cell stages, we analyzed ZAP-70 protein and/or mRNA in normal human B cells at different stages of B cell maturation, including pro/pre-B cells and tumoral cells from 20 B-ALL. ZAP-70 expression was assessed by flow cytometry (FC), immunofluorescence (IF), and/or by quantitative real time RT-PCR (QRT-PCR). In normal bone marrow, ZAP-70 expression was found only in T and in immature B cells (CD19+/CD10+/CD20 −). Moreover, T cells -but no mature B cells- from normal tonsil expressed ZAP-70, as assessed by QRT-PCR and IF. In B-ALLs, a high ZAP-70 expression by FC was observed in 9/13 cases (mean, 82.6%, range 60–99%), whereas in 4 cases ZAP-70 was barely detectable (mean, 13%). By QRT-PCR, 10/16 B-ALLs showed levels of expression similar to ZAP-70 non-expressing cell lines and normal B-cells, whereas in the remaining cases ZAP-70 expression was 3–4 times higher than in normal mature B-cells. Taken together, a high expression of ZAP-70 was found in 11/21 (52%) B-ALLs. No relationship was observed between the level of ZAP-70 expression and the B-ALL maturation status. In conclusion, among normal B cell subsets ZAP-70 expression is restricted to B-cells with pro/pre phenotype. In addition, ZAP-70 is expressed in 52% of B-ALLs, probably as a reflection of their B-cell origin.

Lack of Binding of Monoclonal Protein Produced by Multiple Myeloma (MM) Cells to FcγRIIb Reduces Immunoreceptor Tyrosine-Based Inhibitory Motif (ITIM) Signaling and Provides the Rationale for a Novel Therapeutic Approach to MM

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 455-455 ◽  
Author(s):  
Jennifer Li ◽  
Mingjie Li ◽  
Cathy Wang ◽  
Jessica Wang ◽  
Eric Sanchez ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
M Protein ◽  
Binding Ability ◽  
Monoclonal Protein ◽  
Human B Cells ◽  
Human Sera ◽  
Normal Human ◽  
Time Point

Abstract Abstract 455 Functional B cell and plasma cell immune responses are dependent upon an exquisitely controlled process integrating signals from activating and inhibitory receptors present on the surface of these cells. These activating and inhibitory signaling pathways regulate both the quality and quantity of immunoglobulin (Ig) production. These signaling motifs, termed immunoreceptor tyrosine-based activation motif (ITAM) and ITIM provide the basis for two opposed signaling modules that duel for control of plasma cellular activation within the immune system. The inhibitory Fc receptor FcγRIIb is expressed on plasma cells and controls their persistence in the bone marrow and their ability to produce most serum Ig. Activation of FcγRIIb leads to the phosphorylation of ITIM and recruitment of SH2-containing protein tyrosine phosphatase-1 (SHP-1) SHP-2 and the SH2-containing inositol 5-phosphatase (SHIP) in plasma cells. FcγRIIb-mediated SHIP phosphorylation results in enhanced degradation of PtdIns (3,4,5)P, which is required for B-cell antigen receptor (BCR)-induced recruitment and activation of downstream ITAM signaling molecules. The inhibitory IgG Fc receptor FcγRIIB was the first discovered and remains the best studied example of an ITIM-containing receptor. In this study, we first investigated the IgG-binding ability of 18 MM patients and 10 normal donors to FcγRIIb using flow cytometric analysis. Each serum sample was incubated with MHC1 cells that only express FcγRIIb but do not express FcγRI and FcγRIIa. After washing three times with 1 × PBS, anti-human IgG antibody conjugated with FITC was added to the cells for another 30 minutes. The results showed MM patients' serum IgG have much lower FcγRIIb-binding ability than normal human IgG (P<0.05). Immunohistochemical staining also demonstrated MM patients' sera showed obviously less binding to the FcγRIIb on MHC1 cells compared to serum samples from normal human donors. We further analyzed the FcγRIIb-SHIP signaling pathway in normal B-cells following exposure to MM and normal human sera. Fresh human B-cells were isolated using anti-CD20 antibodies with magnetic bead selection and the cells were washed in an acidic solution (pH 4) for 1 minute to remove any bound antigen from the cell surface. Raji B-cells were also acid-treated. First, these cells were exposed to MM patients' or normal human sera for 5, 15, 30, or 60 minutes to determine the maximum time point of SHIP phosphorylation. Using a standard Western blot protocol, phosphorylated SHIP and total SHIP protein expression were visualized using an enhanced chemiluminescence detection system. The maximum time point of SHIP phosphorylation was 15 minutes. The results showed that both normal human B-cells and Raji B-cells showed markedly lower SHIP phosphorylation/total SHIP following exposure to MM patient serum compared to normal human serum which is consistent with the lack of binding of human Ig to FcγRIIb. Our findings suggest that the monoclonal protein produced by MM patients has very low FcγRIIb-binding ability and is incapable of signaling through inhibitory ITIM pathway. Most importantly, FcγRIIb is expressed on plasma cells and controls their persistence in the bone marrow in addition to Ig production. Cross-linking of FcγRIIb induces apoptosis of plasma cells (Xiang Z et al Nat Immunol 8: 419-29, 2007). Thus, it is possible that this effect prevents the induction of apoptosis in MM cells. Previous studies have reported that normal Ig is capable of inducing B-cell apoptosis. Thus, it is possible that exposure of MM cells to normal immunoglobulin may be capable of inducing apoptosis of these cells and reducing their production of M-protein. We are currently evaluating the ITIM signal transduction pathway, M-protein production and apoptosis in MM and normal B-cells following exposure to normal and MM M-protein IgG. Disclosures: No relevant conflicts of interest to declare.

Progressive Epigenetic Programming during B Cell Maturation Is Reflected in a Continuum of Epigenetic Disease Phenotypes in Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2436-2436 ◽  
Author(s):  
Christopher C. Oakes ◽  
Marc Seifert ◽  
Yassen Assenov ◽  
Lei Gu ◽  
Martina Przekopowitz ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factor ◽  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Malignant Phenotype ◽  
Epigenetic Programming ◽  
B Cell Subsets

Abstract The malignant phenotype combines characteristics that are acquired and inherited from the normal cell of origin. Hematological malignancies and related disease subtypes are thought to arise from diverse cell types that may reflect various developmental stages within the hematopoetic lineage. The contribution of different normal cell states and processes to the biological and clinical features of malignancy is not well understood. In chronic lymphocytic leukemia (CLL), two or three subtypes have been identified by variation in the degree of somatic IGHV mutations and recently uncovered epigenetic differences, respectively, suggesting that these subtypes derive from distinct normal B cell subsets at different stages of maturity. However, in CLL, as well as in most malignancies, the full possible extent of maturity states and the relative contribution of normal versus malignant developmental programs to the malignant phenotype have not been defined in a high-resolution manner. It is widely accepted that epigenetic patterns are important to establish and stabilize cellular phenotypes. Using whole genome bisulfite sequencing and sequence-specific methods, we assessed the dynamic DNA methylation events that occur during the maturation of B cells using six highly purified B cell subsets representing various stages of maturation. We confirmed previous reports that broad epigenetic programming affects about 25% of the genome from naïve to memory B cells, and further revealed that B cell subpopulations of intermediate maturity retained increasing degrees of the maturation program resulting in a singular developmental trajectory. Maturation was driven in part by the activity of a specific set of transcription factors (e.g. AP-1, EBF1, RUNX3, OCT2, IRF4 and NFkB). Using the developmental epigenetic signature defined by transcription factor binding site (TFBS) programming in normal cells to compare to tumor cells of 268 CLL revealed that tumors have the potential to derive from a continuum of possible maturation states that are reflected in the maturation stages of normal cells. Using RNA sequencing to measure gene expression, we found the degree of maturation achieved in tumors closely associates with the acquisition of a more indolent pattern of gene expression, evidenced by progressive downregulation of CLL oncogenes, such as ZAP70, TCL1 and BTK. Further assessment of the level of DNA methylation maturity in an independent sample cohort of 348 CLL cases revealed a quantitative, continuous relationship with increasingly favorable clinical outcomes. Although the majority of methylation differences found between tumor subtypes are naturally present in normal B cells, by identifying changes that are only present in CLL we further uncovered a previously unappreciated pathogenic role of transcription factor dysregulation. Specifically, a blockade in the epigenetic maturation of EBF and AP-1 TFBSs was found to define low-programmed (less mature, poor outcome) CLL cases and was associated with transcriptional and genetic loss of EBF1 and FOS transcription factors in tumor cells. Aberrantly acquired DNA methylation events in CLL were linked to excess activity of specific transcription factor families, namely EGR and NFAT. Intriguingly, we show that recurrent somatic mutations within the DNA binding domain of EGR2 selectively influence the methylation status of its cognate binding sites in mutant cases, establishing a role for this transcription factor in epigenetic dysregulation in CLL. Collectively, this work reveals that a unique epigenetic maturation signature, directed by normal developmental processes, defines individual CLL cases resulting in a spectrum of maturity across tumors. The majority of DNA methylation differences observed between individual CLLs reflects the state of maturity of the founder cell and profoundly influences the disease phenotype. We further propose that in CLL the disease-specific state results, in part, by dysregulation of key transcription factors that imbalance the normal B cell epigenetic program. Disclosures Kipps: Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; AbbVie: Consultancy, Research Funding. Stilgenbauer:AbbVie: Consultancy, Other: travel grants, Research Funding; Amgen: Consultancy, Other: travel grants, Research Funding; Boehringer-Ingelheim: Consultancy, Other: travel grants, Research Funding; Celgene: Consultancy, Other: travel grants, Research Funding; Hoffman-LaRoche: Consultancy, Honoraria, Other: travel grants, Research Funding; Genentech: Consultancy, Other: travel grants, Research Funding; Genzyme: Consultancy, Other: travel grants, Research Funding; Gilead: Consultancy, Other: travel grants, Research Funding; GlaxoSmithKline: Consultancy, Other: travel grants, Research Funding; Janssen: Consultancy, Other: travel grants, Research Funding; Mundipharma: Consultancy, Other: travel grants, Research Funding.

The BCL11 gene family: involvement of BCL11A in lymphoid malignancies

Blood ◽  
2001 ◽  
Vol 98 (12) ◽  
pp. 3413-3420 ◽  
Author(s):  
Ed Satterwhite ◽  
Takashi Sonoki ◽  
Tony G. Willis ◽  
Lana Harder ◽  
Rachael Nowak ◽  
...  
Keyword(s):  
B Cells ◽  
Gene Family ◽  
B Cell ◽  
Family Involvement ◽  
Cpg Island ◽  
Rare Event ◽  
Fetal Brain ◽  
Chromosomal Translocation ◽  
Lymphocytic Leukemia ◽  
Lymphoid Malignancies

Abstract Many malignancies of mature B cells are characterized by chromosomal translocations involving the immunoglobulin heavy chain(IGH) locus on chromosome 14q32.3 and result in deregulated expression of the translocated oncogene. t(2;14)(p13;q32.3) is a rare event in B-cell malignancies. In contrast, gains and amplifications of the same region of chromosome 2p13 have been reported in 20% of extranodal B-cell non-Hodgkin lymphomas (B-NHL), in follicular and mediastinal B-NHL, and in Hodgkin disease (HD). It has been suggested that REL, an NF-κB gene family member, mapping within the amplified region, is the pathologic target. However, by molecular cloning of t(2;14)(p13;q32.3) from 3 cases of aggressive B-cell chronic lymphocytic leukemia (CLL)/immunocytoma, this study has shown clustered breakpoints on chromosome 2p13 immediately upstream of a CpG island located about 300 kb telomeric of REL. This CpG island was associated with a Krüppel zinc finger gene (BCL11A), which is normally expressed at high levels only in fetal brain and in germinal center B-cells. There were 3 major RNA isoforms ofBCL11A, differing in the number of carboxy-terminal zinc fingers. All 3 RNA isoforms were deregulated as a consequence of t(2;14)(p13;q32.3). BCL11A was highly conserved, being 95% identical to mouse, chicken, and Xenopus homologues.BCL11A was also highly homologous to another gene(BCL11B) on chromosome 14q32.1. BCL11Acoamplified with REL in B-NHL cases and HD lymphoma cell lines with gains and amplifications of 2p13, suggesting thatBCL11A may be involved in lymphoid malignancies through either chromosomal translocation or amplification.

scholarly journals Ki-B5: a monoclonal antibody unrelated to CD45 recognizes normal and neoplastic human B cells in routine paraffin sections

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 809-817 ◽  
Author(s):  
ML Hansmann ◽  
HH Wacker ◽  
J Gralla ◽  
H Lumbeck ◽  
M Kossmahl ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Protein Fractions ◽  
Paraffin Sections ◽  
Human B Cells ◽  
Normal Human

In the search for immunoreagents appropriate for the histopathologic diagnosis of malignant B-cell lymphomas in routinely processed paraffin sections, a new monoclonal antibody, Ki-B5, was generated using a high- grade B-cell lymphoma as the immunogene. Ki-B5 is a mouse IgG1/kappa that recognizes five protein fractions of about 84, 82, 55, 48, and 27 Kd after biosynthetic radiolabeling and immunoprecipitation. Protein fractions with the molecular weights of approximately 84 and 82 Kd were expressed on the cell surface and show that Ki-B5 is probably unrelated to CD45. It was possible through electron microscopy to visualize the membrane-bound portion of Ki-B5. Extensive immunohistologic studies on normal human tissue and various neoplasias demonstrated the high specificity of Ki-B5 to normal human B cells and a minor subgroup of plasma cells. Except for ML-2, which is a myelomonocytic human cell line, Ki-B5 exclusively recognized the B-cell lineage, including EB-3, BALL-1, and NALM-1. All carcinomas, sarcomas, and malignant melanomas tested with Ki-B5 were negative. Although normal granulocytes and monocytes were constantly negative, three of eight myelomonocytic leukemias coreacted with this antibody. Eight of the 57 T-cell lymphomas studied were positive to Ki-B5. Five were classified as lymphoblastic, two represented T8-CLL, and one was classified as immunoblastic T-cell lymphoma. Only 3 of 126 cases of B-cell lymphoma, including rare types not considered in the current classifications, were negative to Ki-B5. Plasmacytomas were also negative, except for one case. Irrespective of the cases of lymphoblastic lymphoma and plasmacytoma, Ki-B5 represents a new monoclonal antibody appropriate for the diagnosis and immunophenotyping of malignant lymphomas in routinely processed paraffin sections.

Effects of c-Src Overexpression in CD40-Activated Human B Lymphocytes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3868-3868
Author(s):  
Daniel Jung ◽  
Marie-Pierre Cayer ◽  
Maryse Proulx ◽  
Xue-Zhong Ma ◽  
Darinka Sakac ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Expansion ◽  
Kinase Activity ◽  
Dominant Negative ◽  
Human B Cells ◽  
Normal Human ◽  
Transcriptional Pathway

Abstract The 60-kDa c-Src is the normal human cellular protein counterpart of the highly transforming v-src gene. Recently, c-Src activity was reported to increase in CD40-activated human B cells in the presence of IL-4, suggesting its involvement in proliferation. We report here that c-Src expression is detectable concomitant with the detection of Stat5b and, therefore, Stat5b may serve as a substrate for tyrosine phosphorylation by c-Src, inducing the activation of Stat5b and initiating a transcriptional pathway important for B cell expansion. To elucidate the exact role of c-Src in the proliferation of normal B cells, we undertook c-Src over-expression experiments. Recombinant adenoviruses Ad5/F35 vectors, which we previously reported as highly efficient for B cell transduction, encoding wild-type c-Src(c-Src/WT), constitutively active c-Src(c-Src/CA), dominant negative c-Src(c-Src/DN) or EYFP were constructed. B lymphocytes purified from human peripheral blood were activated with soluble CD154 in the presence or absence of IL-2, IL-4 and IL-10, and infected with the viruses. Real-time PCR and Western blot analysis revealed that vector-transferred c-Src were strongly expressed in infected B cells as early as 48 hours post infection. Kinase assays confirmed that vector-transferred c-Src/WT and c-Src/CA display a strong kinase activity whereas negligible kinase activity was detected with Ad5/F35-c-Src/DN. No significant variation of B cell expansion could be observed between uninfected cells, Ad/F35-EYFP and Ad5/F35-c-Src/CA or Ad5/F35-c-Src/WT infected cells, suggesting that B cell proliferation induced by endogenous c-Src already attains a maximum rate of expansion, which cannot be further enhanced by supplemental exogenous c-Src. In contrast, overexpression of c-Src/DN results in a 40% inhibition of B cell expansion. These results suggest that transgenic dominant negative c-Src may compete with endogenous c-Src resulting in a partial inhibition of a transcriptional pathway involved in B cell proliferation. In conclusion, our results confirm an important role for c-Src in the expansion of normal human B cells in vitro.

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