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

Abstract

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