Stem and progenitor cells are dependent on cytokines for survival and growth; withdrawal of growth factors initiates apoptosis. Understanding the cytokines involved in survival/anti-apoptosis and intracellular mechanisms of action may enhance the efficacy of clinical treatment of disordered hematopoiesis. Our studies with Flt3-ligand (FL), thrombopoietin (TPO), and Stromal Cell Derived Factor (SDF)-1, and those of others, have implicated these cytokines, in survival in vitro of progenitor cells subjected to growth factor withdrawal. We hypothesize that SDF-l, FL and TPO act in concert through common and distinct intracellular signaling pathways and cross-talk to enhance cell survival alone, and synergistically in combination. Towards our long-term goal of obtaining clinical translational information, we propose the following aims: 1) Investigate cellular effects of SDF-1, FL and/or TPO on prolonging survival of primary myeloid progenitor cells (MPC) and growth factor-dependent hematopoietic cell lines that undergo apoptosis upon growth factor withdrawal. Utilize human and murine MPC, as well as MPC from mice expressing an SDF-1 transgene. a) Since mechanisms of cell survival may vary depending on the cytokine(s), cell type and differentiation/maturation and developmental status of cells, assess SDF-1, FL and/or TPO on MPC from different tissues and at different maturation states as determined by cell surface phenotype and responsiveness to stimulation of cell proliferation by single vs. multiple growth factors. b) Since serum contains factors that probably affect kinetics and degree of apoptosis, determine effects of growth factor-withdrawal in the absence and presence of varying concentrations of serum. c) Evaluate survival enhancing effects in MPC overexpressing receptors for these cytokines: CXCR4, Flt3, and C-MPL, after gene transduction. 2) Elucidate intracellular mechanisms involved in anti-apoptotic effects of SDF-l, FL and TPO, on the following pathways: PI3K/Akt, ERK-MAPK/RSK, survivin/p2lc1plPl/Wafl caspases, selected Stats, and G-proteins, and potential cross-talk between these pathways. a) Use growth factor-dependent cell lines for detailed biochemical (protein levels, complex formation, phosphorylation/kinase activities) and multivariate flow cytometric analysis of proteins. Use """"""""dominant-negative"""""""" proteins to assess for functional effects on cell survival. b) Evaluate relevant pathways in primary MPC from humans and mice, including from SDF- 1 transgenic and littermate mice. Use multivariate flow cytometric analysis and overexpression of normal or dominant negative genes for identified signaling molecules. Use MPC from mice functionally-deleted in proposed intracellular signaling molecules to determine dominant effects of these intracellular molecules in survival/anti-apoptotic events.
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