This proposal is to investigate the functions of NR13, a new member of the Bcl-2 family of cell death antagonists, in B cell development and neoplastic transformation by an experimental model system based on the chicken bursa of Fabricius. Preliminary data suggest that NR13 is expressed inversely with programmed cell death in normal and neoplastic bursal B-cells, and that overexpressing NR13 protects DT40, a bursa lymphoma- derived cell line, from serum growth factor withdrawal-induced apoptosis. We have prepared the constructs for NR13 targeted homologous recombination and obtained heterozygous knock-out cells. Attempts to produce homozygous NR13 deletion have failed to yield viable DT40 cells. The heterozygous knock-out cells will be infected with retroviral vector containing NR13 under a tetracycline-regulated promoter, and the attempt to generate homozygous knock-out cells will be repeated to determine whether the viral NR13 transgene will rescue homozygous knock-out cells. If NR13 is essential for DT40 cells survival, we expect NR13 homozygous knock out DT40 cells will only be viable in the absence of tetracycline. This experiment will confirm that NR13 is essential for DT40 bursal lymphoma cell survival. Sequence analysis of NR13 protein suggests that an N-terminal alpha-helix could be a BH4 domain different from that of other Bcl-2 family members and responsible for NR13-specific functions. Expression of deletion mutant for this BH4 domain predisposed DT40 cells to serum withdrawal-induced apoptosis. We propose to utilize the yeast two-hybrid system and affinity purification of NR13 binding proteins to identify the proteins which interact with NR13 through BH4 domain. The specificity of the interaction will be confirmed by the co-immunoprecipitation of in vitro translated interacting protein with NR13 antibody. To study whether NR13 will inhibit bursal cell apoptosis in vivo and facilitate tumor progression, we will construct a bicistronic retroviral vector for co-expressing NR13 and v-myc. The vector will be introduced into our bursa transplant model. The secondary bursa will be analyzed by morphology, apoptosis TUNEL assay and Western analysis for NR13 expression. We plan to attempt to clone NR13 orthologue in mouse and human. The human NR13 orthologue will be used to probe human tumors and normal tissues by Northern analysis to look for up-regulation of NR13 during tumor progression. Information from this chicken lymphoma model could be extended to human to contribute to the understanding the role of cell death regulation in the pathogenesis of human tumors and prognostic significance of NR13.
|Liu, J; Weiss, A; Durrant, D et al. (2004) The cardiolipin-binding domain of Bid affects mitochondrial respiration and enhances cytochrome c release. Apoptosis 9:533-41|
|Lee, R; Chen, J; Matthews, C P et al. (2001) Characterization of NR13-related human cell death regulator, Boo/Diva, in normal and cancer tissues. Biochim Biophys Acta 1520:187-94|