Interferon-gamma (IFN-g) exerts potent inhibitory or stimulatory effects on B lineage cells depending on the stage in B-cell development. This renewal application is aimed at generating molecular genetic profiles of the programs of gene activity in the differential IFN-g responses at two key transitions in B lymphopoiesis: IFN-g-induced growth arrest/apoptosis in early IL-7- and stromal cell-dependent pro-B and pre-B-cells versus IFN-g-stimulated differentiation of late stage pre-B-cells. In comparing two recently developed high throughput gene discovery methods for isolating differentially-expressed genes, the investigator already has identified a set of cDNA clones activated in the IFN-g-induced maturation of late stage 70Z/3 pre-B-cells to surface IgM expression. Half of these are new isolates with no significantly-related matches in DNA sequence databases. The high throughput gene discovery component of these studies is complemented by both minispot and microarray high density gene expression screens aimed at defining the gene programs that are differentially-induced and repressed by IFN-g at different stages in precursor B-cell development. The gene program identified in IFN-g-treated early stage pre-B-cells will also be screened in pro-B and pre-B-cells stimulated to undergo growth arrest/apoptosis by other factors known to inhibit lymphopoiesis (e.g., IFN-alpha, prostaglandin E2, IL-1 alpha glucocorticoids). Gene function studies will focus on those genes shown by gene expression screens to be uniquely expressed in the IFN-g-induced programs of pre-B-cell apoptosis versus differentiation and therefore likely to be involved in controlling these processes. The functions of IFN-g responsive genes will be determined using gene complementation/replacement and anti-sense translation arrest in pro-B and pre-B-cell liens. Targeted ES cell gene knockouts and RAG 2-/- blastocyst complementation will be used to assess gene function in lymphopoiesis. These gene discovery and function studies are expected to reveal how IFN-g induces the differential responses of apoptosis versus differentiation in precursor B-cells. Genes involved in pre-B-cell apoptosis are especially compelling targets for new drug therapies for the treatment of the most common childhood leukemias (i.e., pre-B Acute Lymphoblastic Leukemias).
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