MOLECULAR CHARACTERIZATION OF GM-CSF ACTION

粒细胞-巨噬细胞集落刺激因子（GM-CSF）是一种 促进增殖的造血生长因子， 成熟的骨髓祖细胞和增强其功能。 GM-CSF 有效改善化疗引起的骨髓抑制， 促进骨髓移植后的血液学恢复。 的 GM-CSF的生物活性由高亲和力受体介导， 其由α和β亚基（也与IL-3共享）组成， 和IL-5受体）。 精确的生化和分子事件 介导GM-CSF的作用目前尚不清楚。 我们的实验室已经证明了快速和短暂的诱导， 即早基因Egr-1，在增殖和终末 分化的造血细胞 通过使用Egr-1诱导作为 终点，我们已经开始鉴定介导GM-CSF诱导的 基因表达。 这将使我们能够反向工作，以确定关键 GM-CSF信号转导途径中的步骤。 重组构建体 含有人Egr-1启动子和氯霉素的区域 乙酰转移酶（CAT）报告基因瞬时转染入 GM-CSF或IL-3依赖性细胞系TF-1 . 初步结果 已经证明GM-CSF和IL-3通过重叠和 不同的序列，表明它们的信号通路不同。 该提案的目的是：1）精确识别核苷酸 调节GM-CSF诱导基因的人Egr-1启动子的序列 表达; 2）断裂和表征核因子 与GM-CSF应答序列相互作用;和3）确定 这些因子在正常和肿瘤靶细胞中的作用。 总体而言，这些 调查将提供新的和重要的信息， 控制骨髓细胞增殖的机制， 深入了解骨髓性白血病的病理生理学和治疗。 作为一名儿科血液科医生/肿瘤科医生，我的最终目标是了解 病人的生物学和临床管理之间的关系 有可能致命的疾病 这个项目可以让我 追求我对学术儿科血液学/肿瘤学的兴趣， 弥合基础科学研究与临床医学之间的差距。

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.