Expression and role of the human anti-apoptotic bfl-1 gene in Hodgkin's Lymphoma

Hodgkin's Lymphoma (HL) is identified histologically by the presence of mononuclear Hodgkin (H) cells and multinucleated Reed-Stemberg (RS) cells surrounded by a background of lymphocytes, plasma cells, eosinophils, histocytes and stromal cells in the affected lymph nodes. HL affects on average 2.5 per 100,000 of population and between 30 and 50 % of HL cases are Epstein-Barr virus associated (EBV positive HL). An important feature of HL is the constitutive activation of the NF-kB anscription factor, which has proliferative and anti-apoptotic roles in HIRS cells. Bfl-1 is an anti-apoptotic protein of the Bcl-2 family, whose preferential expression in hematopoietic and endothelial cells is controlled by inflammatory stimuli. This thesis presents the novel finding that bfl-1 is highly expressed in H/RS cells from primary tumour tissue from HL patients and cultured H/RS cells irrespective of their EBV status and that the short splice variant of bfl-I, designated bfl-IS is not expressed in these cell types or in type I Burkitt Lymphoma (BL), type I11 BL or lymphoblastoid cell lines. This thesis presents the novel discovery that bfl-I is a key NF-KB target gene in WRS cells and death by apoptosis caused by inhibition of NF-KB is coincident with a loss of bfl-1 expression. It is shown in this study for the first time that ectopic expression of Bfl-1 protected WRS cells from apoptosis induced by NF-KB inhibition. The downregulation of bjZ-1 in a HL-derived cell line by RNA interference is reported in this study and shown to potentiate the effects of cytotoxic agents by decreasing the cellular apoptotic threshold. The novel finding that NF-KB regulates the bfl-1 promoter, with a key role in this cell context for a novel NF-KB binding site in the upstream regulatory regon of this gene is reported here. This study reports the mechanisms by which bjl-I expression is regulated in WRS cells and serves to establish the contribution of bjZ-1 to the pathogenesis of HL. Also as part of this thesis, as a means to generate an antibody to Bfl-I, a novel vector (pGSLink) was designed and constructed to permit high-level expression of a protein linked at the C- or N-terminal to a His-tag by a flexible, poorly immunogenic linker peptide of 21 amino acids; (Gly4Ser)4Gly (published in Analytical Biochemistry; Loughran et al., 2006). The bfl-1 coding fragment was successfully cloned into this novel vector to produce the fusion constructs from which His-Linker-Bfl-1 fusion proteins were s~rccessfullyo verexpressed and purified using Zmmobilised metal affinity chrornatwgraphy. Sufficient protein was purified and used for the prepantion of polyclonal antisera to Bf1-1. ln summary, Ihe findings presented in this thesis are relevant to our understanding of the role d 6fI-l as a crucial pro-survival NF-KB target in HRS cells and the potential of Bfl-I as a rational thcrapeuric target in HL is highlighted

Expression and role of the human anti-apoptotic bfl-1 gene in Hodgkin\u27s Lymphoma

Hodgkin\u27s Lymphoma (HL) is identified histologically by the presence of mononuclear Hodgkin (H) cells and multinucleated Reed-Stemberg (RS) cells surrounded by a background of lymphocytes, plasma cells, eosinophils, histocytes and stromal cells in the affected lymph nodes. HL affects on average 2.5 per 100,000 of population and between 30 and 50 % of HL cases are Epstein-Barr virus associated (EBV positive HL). An important feature of HL is the constitutive activation of the NF-kB anscription factor, which has proliferative and anti-apoptotic roles in HIRS cells. Bfl-1 is an anti-apoptotic protein of the Bcl-2 family, whose preferential expression in hematopoietic and endothelial cells is controlled by inflammatory stimuli. This thesis presents the novel finding that bfl-1 is highly expressed in H/RS cells from primary tumour tissue from HL patients and cultured H/RS cells irrespective of their EBV status and that the short splice variant of bfl-I, designated bfl-IS is not expressed in these cell types or in type I Burkitt Lymphoma (BL), type I11 BL or lymphoblastoid cell lines. This thesis presents the novel discovery that bfl-I is a key NF-KB target gene in WRS cells and death by apoptosis caused by inhibition of NF-KB is coincident with a loss of bfl-1 expression. It is shown in this study for the first time that ectopic expression of Bfl-1 protected WRS cells from apoptosis induced by NF-KB inhibition. The downregulation of bjZ-1 in a HL-derived cell line by RNA interference is reported in this study and shown to potentiate the effects of cytotoxic agents by decreasing the cellular apoptotic threshold. The novel finding that NF-KB regulates the bfl-1 promoter, with a key role in this cell context for a novel NF-KB binding site in the upstream regulatory regon of this gene is reported here. This study reports the mechanisms by which bjl-I expression is regulated in WRS cells and serves to establish the contribution of bjZ-1 to the pathogenesis of HL. Also as part of this thesis, as a means to generate an antibody to Bfl-I, a novel vector (pGSLink) was designed and constructed to permit high-level expression of a protein linked at the C- or N-terminal to a His-tag by a flexible, poorly immunogenic linker peptide of 21 amino acids; (Gly4Ser)4Gly (published in Analytical Biochemistry; Loughran et al., 2006). The bfl-1 coding fragment was successfully cloned into this novel vector to produce the fusion constructs from which His-Linker-Bfl-1 fusion proteins were s~rccessfullyo verexpressed and purified using Zmmobilised metal affinity chrornatwgraphy. Sufficient protein was purified and used for the prepantion of polyclonal antisera to Bf1-1. ln summary, Ihe findings presented in this thesis are relevant to our understanding of the role d 6fI-l as a crucial pro-survival NF-KB target in HRS cells and the potential of Bfl-I as a rational thcrapeuric target in HL is highlighted

Nuclear Factor κB-Dependent Activation of the Antiapoptotic bfl-1 Gene by the Epstein-Barr Virus Latent Membrane Protein 1 and Activated CD40 Receptor

Suppression of the cellular apoptotic program by the oncogenic herpesvirus Epstein-Barr virus (EBV) is central to both the establishment of latent infection and the development of EBV-associated malignancies. We have previously shown that expression of the EBV latent membrane protein 1 (LMP1) in Burkitt's lymphoma cell lines leads to increased mRNA levels from the cellular antiapoptotic bfl-1 gene (also known as A1). Furthermore, ectopic expression of Bfl-1 in an EBV-positive cell line exhibiting a latency type 1 infection protects against apoptosis induced by growth factor deprivation (B. N. D'Souza, M. Rowe, and D. Walls, J. Virol. 74:6652-6658, 2000). We now report that LMP1 drives bfl-1 promoter activity through interactions with components of the tumor necrosis factor receptor (TNFR)/CD40 signaling pathway. We present evidence that this process is NF-κB dependent, involves the recruitment of TNFR-associated factor 2, and is mediated to a greater extent by the carboxyl-terminal activating region 2 (CTAR2) relative to the CTAR1 domain of LMP1. Activation of CD40 receptor also led to increased bfl-1 mRNA levels and an NF-κB-dependent increase in bfl-1 promoter activity in Burkitt's lymphoma-derived cell lines. We have delineated a 95-bp region of the promoter that functions as an LMP1-dependent transcriptional enhancer in this cellular context. This sequence contains a novel NF-κB-like binding motif that is essential for transactivation of bfl-1 by LMP1, CD40, and the NF-κB subunit protein p65. These findings highlight the role of LMP1 as a mediator of EBV-host cell interactions and may indicate an important route by which it exerts its cellular growth transforming properties

Repression of the proapoptotic cellular BIK/NBK gene by Epstein-Barr virus antagonizes transforming growth factor β1-induced B-cell apoptosis

The Epstein-Barr virus (EBV) establishes a lifelong latent infection in humans. EBV infection of primary B cells causes cell activation and proliferation, a process driven by the viral latency III gene expression program, which includes EBV nuclear proteins (EBNAs), latent membrane proteins, and untranslated RNAs, including microRNAs. Some latently infected cells enter the long-lived memory B-cell compartment and express only EBNA1 transiently (Lat I) or no EBV protein at all (Lat 0). Targeting the molecular machinery that controls B-cell fate decisions, including the Bcl-2 family of apoptosis-regulating proteins, is crucial to the EBV cycle of infection. Here, we show that BIK (also known as NBK), which encodes a proapoptotic “sensitizer” protein, is repressed by the EBNA2-driven Lat III program but not the Lat I program. BIK repression occurred soon after infection of primary B cells by EBV but not by a recombinant EBV in which the EBNA2 gene had been knocked out. Ectopic BIK induced apoptosis in Lat III cells by a mechanism dependent on its BH3 domain and the activation of caspases. We show that EBNA2 represses BIK in EBV-negative B-cell lymphoma-derived cell lines and that this host-virus interaction can inhibit the proapoptotic effect of transforming growth factor β1 (TGF-β1), a key physiological mediator of B-cell homeostasis. Reduced levels of TGF-β1-associated regulatory SMAD proteins were bound to the BIK promoter in response to EBV Lat III or ectopic EBNA2. These data are evidence of an additional mechanism used by EBV to promote B-cell survival, namely, the transcriptional repression of the BH3-only sensitizer BIK. IMPORTANCE Over 90% of adult humans are infected with the Epstein-Barr virus (EBV). EBV establishes a lifelong silent infection, with its DNA residing in small numbers of blood B cells that are a reservoir from which low-level virus reactivation and shedding in saliva intermittently occur. Importantly, EBV DNA is found in some B-cell-derived tumors in which viral genes play a key role in tumor cell emergence and progression. Here, we report for the first time that EBV can shut off a B-cell gene called BIK. When activated by a molecular signal called transforming growth factor β1 (TGF-β1), BIK plays an important role in killing unwanted B cells, including those infected by viruses. We describe the key EBV–B-cell molecular interactions that lead to BIK shutoff. These findings further our knowledge of how EBV prevents the death of its host cell during infection. They are also relevant to certain posttransplant lymphomas where unregulated cell growth is caused by EBV genes