MECHANISMS OF SYNERGISTIC REGULATION OF STEM/PROGENITORS

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Cytokines act in vitro to stimulate, enhance, and/or suppress proliferation of myeloid progenitor cells (MPC). Actions of some cytokines noted in vitro have been reproduced in animal models, and some have shown efficacy in human clinical trials. It is probable that synergism noted in vitro in terms of stimulation of MPC proliferation in response to combinations of cytokines, especially a potent co-stimulating cytokine such as steel factor, and suppression of this synergism by chemokines are of relevance to hematopoiesis in vivo. It is our belief that an evaluation of intracellular mechanisms triggered by these growth promoting and suppressing cytokines will enhance our understanding of normal MPC regulation, and will ultimately be of use in designing rational treatments for patients with hematological disorders. We believe that multi-growth factor induced synergistic stimulation of MPC proliferation and this inhibition by chemokines is, at least in part, a cell cycle phenomenon. Our hypothesis is that cell cycle regulators such as cyclin-dependent kinase inhibitor, p21cip1/waf1, certain cyclin-dependent kinases, and cyclins, and other intracellular signals are key to responses mediating proliferative synergy. Our biochemical results and evaluation of MPC proliferation from mice functionally deleted in some of these proposed intracellular mediators support this hypothesis. Our long-term goal is to define key intracellular molecules involved in these effects and to utilize this information for clinical benefit. Towards these goals we propose the following two Specific Aims: 1. Investigate intracellular mechanisms involved in multiple growth factor (GM-CSF and steel factor)-induced synergistic stimulation of the proliferation of MPC by: a) assessing a potential role for the cyclin-dependent kinase inhibitor/modulator p21cip1/waf1, and its relationship with cyclin-dependent kinases and cyclins in these effects and b) elucidating a potential role for the transcription factors BCL-6 and Stat4, the protein phosphatases SHP-1 and SHIP, and MAP kinase, and their possible interrelationship(s) with each other and with p21cip1/waf1 in these effects. 2. Evaluate intracellular mechanisms involved in suppression of multiple growth factor-induced synergistic stimulation of the proliferation of MPC by assessing comparative effects of myelosuppressive CC (MIP-1alpha, MCP-1), CXC (IL-8, IP-10), and C (lymphotactin) chemokines and the receptors they act on through: a) the cell cycle and regulatory molecules which control cell cycle progression and b) Intracellular signaling molecules linked to stimulation of MPC proliferation.