Regulation Of B-cell Survival Research Articles

The B lineage transcription factor E2A regulates apoptosis in chronic lymphocytic leukemia (CLL) cells

Chronic lymphocytic leukemia (CLL) is a common malignancy characterized by the accumulation of B lymphocytes with an antigen-experienced activated CD19(+)CD5(+) clonal phenotype. Clinically, ∼50% of cases will behave more aggressively. Here, we investigate the role of the major B-cell transcription factor E2A, a known regulator of B-cell survival and proliferation, to CLL persistence. We show that E2A is elevated at the mRNA and protein levels relative to normal B-cell subsets. E2A silencing in primary CLL cells leads to a significant increase in spontaneous apoptosis in both CD38(+) (aggressive) and CD38(-) (indolent) cases. Moreover, E2A knockdown synergizes with the immunomodulatory drug lenalidomide to reduce CLL viability. E2A is known to restrain the proliferation of primary B and T lymphocytes at multiple stages of maturation and we report that targeted E2A disruption increases the frequency of Ki-67(+) CLL cells in the absence of effects on de novo proliferation. At the molecular level, E2A siRNA-treated CLL cells display reduced expression of key genes associated with survival and cell cycling including p27, p21 and mcl-1, of which the former two are known E2A target genes. Thus, E2A, a key transcription factor associated with the B-cell activation profile, regulates apoptosis in CLL and may contribute to disease pathology.

Impaired expression of p66Shc, a novel regulator of B-cell survival, in chronic lymphocytic leukemia

AbstractIntrinsic apoptosis defects underlie to a large extent the extended survival of malignant B cells in chronic lymphocytic leukemia (CLL). Here, we show that the Shc family adapter p66Shc uncouples the B-cell receptor (BCR) from the Erk- and Akt-dependent survival pathways, thereby enhancing B-cell apoptosis. p66Shc expression was found to be profoundly impaired in CLL B cells compared with normal peripheral B cells. Moreover, significant differences in p66Shc expression were observed in patients with favorable or unfavorable prognosis, based on the mutational status of IGHV genes, with the lowest expression in the unfavorable prognosis group. Analysis of the expression of genes implicated in apoptosis defects of CLL showed an alteration in the balance of proapoptotic and antiapoptotic members of the Bcl-2 family in patients with CLL. Reconstitution experiments in CLL B cells, together with data obtained on B cells from p66Shc−/− mice, showed that p66Shc expression correlates with a bias in the Bcl-2 family toward proapoptotic members. The data identify p66Shc as a novel regulator of B-cell apoptosis which attenuates BCR-dependent survival signals and modulates Bcl-2 family expression. They moreover provide evidence that the p66Shc expression defect in CLL B cells may be causal to the imbalance toward the antiapoptotic Bcl-2 family members in these cells.

Impaired Expression of p66Shc, a Novel Regulator of B-Cell Survival, in Chronic Lymphocytic Leukemia.

Abstract 801Intrinsic defects in the apoptotic circuitry underlie to a large extent the extended survival of malignant B cells in chronic lymphocytic leukemia (CLL) and are moreover believed to be responsible for their resistance to chemotherapy. We have recently demonstrated that p66Shc, a member of Shc family of protein adapters, acts as a promoter of apoptosis in T cells. Here we show that p66Shc uncouples the B-cell antigen receptor (BCR) from the Erk and Akt dependent survival pathways, thereby enhancing B-cell apoptosis. Expression of p66Shc was found to be profoundly and consistently impaired in CLL B cells compared to peripheral blood B cells form healthy donors. Moreover, significant differences in p66Shc expression were observed in patients with favorable or unfavorable prognosis, classified on the basis of the mutational status of the IGHV genes, with the lowest expression in the unfavorable prognosis group. Analysis of the expression of genes previously implicated in the apoptosis defects of CLL B cells revealed a selective alteration in the balance of pro- and anti-apoptotic members of the Bcl-2 family in these patients. Reconstitution experiments in CLL B cells, as well as data obtained on B cells from p66Shc-/- mice, showed that p66Shc expression correlates with a bias in the Bcl-2 family towards the pro-apoptotic members. Collectively, the data identify p66Shc as a novel regulator of B cell apoptosis which attenuates survival signals emanating from the BCR and modulates expression of the Bcl-2 family. They moreover provide evidence that the defect in p66Shc expression identified in CLL B cells may be causally related to the imbalance towards the anti-apoptotic Bcl-2 family members observed in these cells. Disclosures:No relevant conflicts of interest to declare.

Nuclear PKCδ Is Deadly

Previously, this group reported that mice deficient for protein kinase Cδ (PKCδ) exhibit a lupus-like autoimmune disease, which is typically caused by proliferation of autoreactive B cells. Overexpression of BAFF (B cell-activating factor belonging to the tumor necrosis factor family) also causes a similar phenotype in mice. Now Mecklenbräuker et al . show that PKCδ deficiency results in mice that do not show decreased B cell numbers or maturation in response to injection of a BAFF decoy. Furthermore, PKCδ-deficient mice that were also expressing a signaling-deficient form of the BAFF receptor did not show reduced sizes in lymphoid organs or fewer peripheral B cells that are typical of mice in which BAFF signaling is blocked. Isolated PKCδ −/− B cells showed increased life-span in culture compared to that of wild-type B cells, and this effect was not due to increased production of the survival factor interleukin 6 (IL-6). Furthermore, PKCδ deficiency did not rescue B cells from apoptosis caused by Btk deficiency (Btk stands for Bruton's tyrosine kinase and is part of an antiapoptotic signaling cascade). Isolated splenic B cells from wild-type mice were susceptible to cell death and exhibited an increase in nuclear PKCδ 24 hours after exposure to serum-containing medium. Exposure of isolated B cells to BAFF prevented nuclear accumulation of PKCδ, and overexpression of PKC resulted in an increase in the abundance of phosphorylated histone 2B at serine 14 (S14-H2B), a phosphorylation event known to be associated with cell death. PKCδ-deficient B cells exhibited decreased abundance of S14-H2b compared with wild-type B cells. Thus, BAFF can mediate cell survival by inhibiting nuclear PKCδ activity. I. Mecklenbräuker, S. L. Kalled, M. Leitges, F. Mackay, A. Tarakhovsky, Regulation of B-cell survival by BAFF-dependent PKCδ-mediated nuclear signalling. Nature 431 , 456-461 (2004). [Online Journal]

Regulation of B-cell survival by BAFF-dependent PKCdelta-mediated nuclear signalling.

Approximately 65% of B cells generated in human bone marrow are potentially harmful autoreactive B cells. Most of these cells are clonally deleted in the bone marrow, while those autoreactive B cells that escape to the periphery are anergized or perish before becoming mature B cells. Escape of self-reactive B cells from tolerance permits production of pathogenic auto-antibodies; recent studies suggest that extended B lymphocyte survival is a cause of autoimmune disease in mice and humans. Here we report a mechanism for the regulation of peripheral B-cell survival by serine/threonine protein kinase Cdelta (PKCdelta): spontaneous death of resting B cells is regulated by nuclear localization of PKCdelta that contributes to phosphorylation of histone H2B at serine 14 (S14-H2B). We show that treatment of B cells with the potent B-cell survival factor BAFF ('B-cell-activating factor belonging to the TNF family') prevents nuclear accumulation of PKCdelta. Our data suggest the existence of a previously unknown BAFF-induced and PKCdelta-mediated nuclear signalling pathway which regulates B-cell survival.

A-myb rescues murine B-cell lymphomas from IgM-receptor–mediated apoptosis through c-myctranscriptional regulation

A-myb is a member of the myb family of transcription factors, which regulates proliferation, differentiation, and apoptosis of hematopoietic cells. A-Myb expression is normally restricted to the proliferating B-cell centroblasts and transgenic mice overexpressing A-myb displayed enhanced hyperplasia of the lymph nodes. Because A-Myb is highly expressed in several subtypes of human B-cell neoplasias, we sought to determine whether the A-myb gene promoted proliferation and survival of B lymphocytes, using the WEHI 231 and CH33 murine B-cell lymphomas as models. Here, we show that ectopic expression of A-mybrescues WEHI 231 and CH33 cells from growth arrest and apoptosis induced by anti-IgM treatment. Previously, we demonstrated an essential role of the c-myc gene in promoting cell survival of WEHI 231 cells in response to a variety of apoptotic stimuli. Furthermore, we and others have shown that the c-myc gene is potently transactivated by A-Myb in several cell types. Thus, we sought to determine whether c-Myc would mediate the A-Myb antiapoptotic effect in B cells. Here we show that ectopic expression of A-myb leads to maintenance of c-myc expression, and that expression of antisense c-myc RNA ablates A-Myb–mediated survival signals. Thus, these findings strongly implicate the A-myb gene in the regulation of B-cell survival and confirm the c-myc gene as one of the downstream targets of A-myb in these cells. Overall, our observation suggests that A-mybexpression may be relevant to the pathology of human B-cell neoplasias.

A-myb rescues murine B-cell lymphomas from IgM-receptor–mediated apoptosis through c-myctranscriptional regulation

AbstractA-myb is a member of the myb family of transcription factors, which regulates proliferation, differentiation, and apoptosis of hematopoietic cells. A-Myb expression is normally restricted to the proliferating B-cell centroblasts and transgenic mice overexpressing A-myb displayed enhanced hyperplasia of the lymph nodes. Because A-Myb is highly expressed in several subtypes of human B-cell neoplasias, we sought to determine whether the A-myb gene promoted proliferation and survival of B lymphocytes, using the WEHI 231 and CH33 murine B-cell lymphomas as models. Here, we show that ectopic expression of A-mybrescues WEHI 231 and CH33 cells from growth arrest and apoptosis induced by anti-IgM treatment. Previously, we demonstrated an essential role of the c-myc gene in promoting cell survival of WEHI 231 cells in response to a variety of apoptotic stimuli. Furthermore, we and others have shown that the c-myc gene is potently transactivated by A-Myb in several cell types. Thus, we sought to determine whether c-Myc would mediate the A-Myb antiapoptotic effect in B cells. Here we show that ectopic expression of A-myb leads to maintenance of c-myc expression, and that expression of antisense c-myc RNA ablates A-Myb–mediated survival signals. Thus, these findings strongly implicate the A-myb gene in the regulation of B-cell survival and confirm the c-myc gene as one of the downstream targets of A-myb in these cells. Overall, our observation suggests that A-mybexpression may be relevant to the pathology of human B-cell neoplasias.

Disregulation of leukosialin (CD43, Ly48, sialophorin) expression in the B-cell lineage of transgenic mice increases splenic B-cell number and survival.

Leukosialin (also known as Ly48, CD43, and sialophorin) is a major cell surface sialoglycoprotein found on a variety of hematopoietically derived cells. The precise function of this molecule is poorly understood but it has been implicated in cell proliferation and intercellular adhesion. We developed a transgenic mouse model to assess leukosialin's function in vivo. Our approach was to alter mouse CD43 (mCD43) expression in the B-cell lineage where it is tightly regulated, by expressing it in peripheral B cells where it is normally absent. To drive expression of leukosialin in mature B cells, the immunoglobulin heavy chain enhancer was fused to the mCD43 gene. mCD43-immunoglobulin heavy chain enhancer transgenic mice display splenomegaly due to increased numbers of B cells. Transgenic B cells show a striking increase in their ability to survive in vitro compared to B cells from nontransgenic control mice. This prolonged survival is reflected in a decreased susceptibility to apoptosis. These observations suggest that mCD43 plays an important role in the regulation of B-cell survival. The alteration of the temporal expression, or "disregulation," of a gene in transgenic mice provides a general strategy for elucidating the in vivo role of other molecules involved in cell signaling and adhesion.