MOLECULAR CHARACTERIZATION OF GM-CSF ACTION

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Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor which promotes the proliferation and maturation of myeloid progenitors and enhances their function. GM-CSF is effective in ameliorating chemotherapy-induced myelosuppression and enhances hematologic recovery following bone marrow transplantation. The biological activity of GM-CSF is mediated by a high-affinity receptor, which consists of an alpha and beta subunit (also shared with the IL-3 and IL-5 receptors). The precise biochemical and molecular events mediating the effects of GM-CSF are presently unknown. Our laboratory has demonstrated the rapid and transient induction of the immediate early gene, Egr-1, in both proliferating and terminally differentiated hematopoietic cells. By using Egr-1 induction as an endpoint, we have begun to identify sequences mediating GM-CSF-induced gene expression. This will allow us to work backwards to identify key steps in the GM-CSF signal transduction pathway. Recombinant constructs containing regions of the human Egr-1 promoter and chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into the GM-CSF- or IL-3-dependent cell line, TF-1 . Preliminary results have demonstrated that GM-CSF and IL-3 work through both overlapping and distinct sequences, suggesting that their signaling pathways diverge. The aims of this proposal are to: 1) precisely identify the nucleotide sequences of the human Egr-1 promoter regulating GM-CSF-induced gene expression; 2) fractionate and characterize the nuclear factors interacting with GM-CSF-responsive sequences; and 3) determine the role of these factors in normal and neoplastic target cells. Overall, these investigations will provide new and important information on the precise mechanisms controlling proliferation of myeloid cells and will yield insights into the pathophysiology and treatment of myeloid leukemias. As a Pediatric Hematologist/Oncologist, my ultimate goal is to understand the relationship between the biology and clinical management of patients who have this potentially fatal condition. This project will allow me to pursue my interests in academic pediatric hematology/oncology and to bridge the gap between basic science research and clinical medicine.