The focus of this project is, a deeper understanding of the early development of the B-cell lineage in the bursa of Fabricius, and the perturbation of that development during induction of malignant lymphomas in the bursa by deregulated expression of the c-myc oncogene. Using a variety of experimental approaches including retroviral vector-mediated gene transfer into bursal stem cells and bursal transplantation, targeted gene deletion/replacement in a bursal lymphoma-derived cell line (DT-40) and microarray analysis, we propose to focus on three groups of specific aims. First our chicken immune system microarray will be expanded, and modifications made to our retroviral vectors in order to detect changes in gene expression associated with progression from preneoplastic to fully neoplastic stages of lymphomagenesis. With this approach the hypothesis that myb-induced bursal lymphomas involve deregulation of c-myc will be tested. Expression profiles of myc-, myb- and v-rel-induced bursal lymphomas will be compared to seek evidence of a common pathway of lymphomagenesis. We will explore the role of the novel anti-apoptotic regulator Nr-13, and the pro-apoptotic protein Mtd/Bok. These relatively little-investigated genes are candidates to be major regulators of the intense apoptosis which accompanies normal B-cell development in the bursa and which is significantly modified during myc-induced neoplastic development. Therefore, second, DT-40 cells deleted for Nr-13 will be analyzed to determine whether it is essential for viability, and a mutational analysis will be carried out to identify domains required for several functions of Nr-13 including the ability of Nr-13 to maintain bursal stem cell function in vivo. Third, We will determine the intracellular localization of two naturally occurring forms of Mtd/Bok, and define the anti-apoptotic proteins with which it/they interact. Testing of the effects of constitutive overexpression of these proteins in bursal stem cell on normal and neoplastic B-cell development will reveal whether a block to apoptosis induced by Mtd/Bok exists and plays a role in tumor progression. Finally dominant inhibitors of Mtd/Bok function will be tested probe the role of this gene in vivo.
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