MECHANISMS OF G-CSF RECEPTOR MEDIATED DIFFERENTIATION

G-CSF受体介导的分化机制

• 批准号: 2668054
• 负责人: David John Tweardy
• 金额: $ 23.9万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2668054
• 关键词: DNA binding protein; acute myelogenous leukemia; biological signal transduction; cell cycle; cell differentiation; cell growth regulation; clinical research; colony stimulating factor; complementary DNA; cytogenetics; growth factor receptors; human subject; immunoprecipitation; laboratory rabbit; myeloid stem cell; neoplastic cell; neutrophil; northern blottings; polymerase chain reaction; prognosis; protein tyrosine kinase; receptor binding; tissue /cell culture; transcription factor; western blottings

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.

描述：(改编自研究人员摘要)G-CSF是 驱动中性粒细胞的最关键的细胞因子 差异化。大多数AML病例都来自这一血统。 然而，AML细胞对G-CSF的反应异常，在没有G-CSF的情况下增殖 差异化。这种异常反应的基础尚不清楚。 表征G-CSF信号转导途径的初步尝试 应用传统生化技术在正常和白血病髓系细胞系中的应用 研究没有得出多少有用的信息作为比较的基础。 正常髓系细胞和白血病髓系细胞之间。调查人员已经 确定了三种磷酸蛋白-pp55、pp70和pp80-是 激活并与G-CSFR关联。调查人员已经 确定pp55是Src相关蛋白酪氨酸激酶(PTK)， Lyn，而pp70是与Src无关的PTK，Syk。调查人员已经 发现了一个潜在的基于酪氨酸的激活基序()，它可能 在胞浆的远端半部内作为Syk对接部位 受体的结构域区被证明是必需的和特异的 中性粒细胞分化。调查人员的初步研究 支持pp80可能是信号的新成员的假设 转导和转录激活因子(STAT)蛋白家族， 指定的统计数据。研究人员证明，激活 STATG与增殖无关；相反，最佳激活 需要G-CSFR的特定于差异的域，该域包含 推定的赛克对接地点。在所有测试的正常人类髓系细胞中 (包括CD34+骨髓细胞)，G-CSF激活DNA结合 复合体，G-SIF-A，仅由STATG组成。与普通人不同 7个人AML细胞系中6个和5个AML细胞系中的髓系细胞G-SIF-A 12例AML患者样本同时含有StatG和STAT3。这些 研究结果表明，替代STAT蛋白的激活 某些AML细胞中G-CSF表达的STAT3可能是导致其不能 遵循正常的G-CSF激活的分化程序。这一效果 可能通过STAT3与StatG结合的竞争来调节 反式激活分化特异的启动子元件 基因。 这项提议的具体目标是：目标一、纯化和克隆 STATG及其与G-CSFR相互作用的分子表征 在正常的髓系细胞中。目标II.描述Syk的交互作用 G-CSFR在正常髓系细胞中的表达并确定其在 STATG的激活、分化和增殖。目标III.至 急性髓系白血病患者样本中G-SIF-A的特征及其与 G-SIF-A(单独STATG与STATG+STAT3)的生物学组成 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。的长期目标 这项提议是为了找出导致 急性髓系白血病细胞对粒细胞集落刺激因子的异常反应 旨在克服这些异常的分化疗法。