MECHANISMS OF G-CSF RECEPTOR MEDIATED DIFFERENTIATION

描述：（改编自研究者摘要）G-CSF是一种 最关键的细胞因子驱动嗜中性粒细胞 分化大多数AML病例都来自这一谱系。 然而，AML细胞对G-CSF的异常反应是增殖，而不 差异化这种异常反应的基础尚不清楚。 G-CSF信号转导通路的初步研究 在正常和白血病骨髓细胞系中，使用传统的生物化学 研究没有提供多少有用的信息作为比较的基础 正常骨髓细胞和白血病骨髓细胞的区别调查人员已 鉴定了三种磷蛋白-pp 55，pp 70和pp 80， 激活并与G-CSFR相关联。调查人员已 确定pp 55是Src相关蛋白酪氨酸激酶（PTK）， 林恩，而pp 70是非Src相关的PTK，Syk。调查人员已 鉴定了一种潜在的基于酪氨酸的激活基序（TAM）， 作为Syk对接位点，位于细胞质的远侧半部分， 受体的结构域区域显示是必需的和特异性的， 嗜酸性分化调查人员的初步研究 支持pp 80可能是信号的新成员的假设 转录转导子和激活子（STAT）蛋白家族， 命名为StatG。研究人员证明， StatG与增殖无关;相反， 需要G-CSFR的差异化特定域， 假定的赛克对接点在所有测试的正常人类骨髓细胞中 （包括CD 34+骨髓细胞），G-CSF激活DNA结合 复合物，G-SIF-A，仅由StatG组成。与正常人相比， 骨髓细胞，G-SIF-A在七个人AML细胞系中的六个和 12个AML患者样品含有StatG和Stat 3。这些 研究结果表明，激活替代STAT蛋白， 在某些AML细胞中，G-CSF中的Stat 3可能导致它们不能通过G-CSF表达。 遵循正常的G-CSF激活分化程序。这种效果 可能是通过Stat 3竞争StatG与 反式激活分化特异性 基因. 该提案的具体目标是：目标一。提纯和克隆 StatG和分子表征其与G-CSFR的相互作用 在正常的骨髓细胞中。AIM II.为了描述赛科与 G-CSFR在正常骨髓细胞中的作用，并确定其在 StatG活化、分化和增殖。AIM III.到 表征AML患者样品中的G-SIF-A，并将其与AML患者样品中的G-SIF-A相关联。 G-SIF-A（单独StatG与StatG + Stat 3）与生物制剂的组合物 细胞对G-CSF的反应，细胞遗传学特征，表面 免疫表型、FAB亚类和预后。长期目标是 这项建议是为了确定说明 AML细胞对G-CSF的异常反应，以设计合理的 针对这些异常的分化疗法。

DESCRIPTION: (Adapted from investigator's abstract) G-CSF is one of the most critical cytokines driving neutrophilic granulocyte differentiation. The majority of AML cases arise from this lineage. However, AML cells respond aberrantly to G-CSF by proliferating without differentiating. The basis for this aberrant response is unknown. Initial attempts to characterize the G-CSF signal transduction pathway in normal and leukemic myeloid cell lines using traditional biochemical studies yielded little information useful as a basis for comparison between normal and leukemic myeloid cells. The investigators have identified three phosphoproteins - pp55, pp70, and pp80 - that are activated and associate with the G-CSFR. The investigators have determined that pp55 is the Src-related protein tyrosine kinase (PTK), Lyn, while pp70 is the non-Src-related PTK, Syk. The investigators have identified a potential tyrosine-based activation motif (TAM) that may serve as a Syk docking site within the distal half of the cytosolic domain region of the receptor shown to be essential and specific for neutrophilic differentiation. The investigators' preliminary studies support the hypothesis that pp80 may be a novel member of the signal transducers and activators of transcription (STAT) protein family, designated StatG. The investigators demonstrated that activation of StatG did not correlate with proliferation; rather, optimal activation requires the differentiation-specific domain of the G-CSFR that contains the putative Syk docking site. In all normal human myeloid cells tested (including CD34+ bone marrow cells), G-CSF activated a DNA-binding complex, G-SIF-A, composed solely of StatG. In contrast to normal human myeloid cells, G-SIF-A in six of seven human AML cell lines and five of twelve AML patient samples contained both StatG and Stat3. These findings suggests that activation of alternative STAT proteins such as Stat3 by G-CSF in some AML cells may contribute to their failure to follow the normal G-CSF-activated differentiation program. This effect may be mediated through competition by Stat3 for StatG binding to promoter elements critical for transactivating differentiation-specific genes. The Specific Aims of this proposal are: AIM I. To purify and clone StatG and to molecularly characterize its interaction with the G-CSFR in normal myeloid cells. AIM II. To characterize Syk's interaction with the G-CSFR in normal myeloid cells and to determine its role in StatG activation, differentiation, and proliferation. AIM III. To characterize G-SIF-A in AML patient samples and to correlate the composition of G-SIF-A (StatG alone vs. StatG + Stat3) with the biologic response of cells to G-CSF, cytogenetic features, surface immunophenotype, FAB subclass, and prognosis. The long range goal of this proposal is to identify the mechanisms that account for the aberrant response to G-CSF in AML cells in order to design rational differentiation therapies targeted to overcome these abnormalities.