MECHANISMS OF MURINE CFU-E MATURATION

小鼠 CFU-E 成熟机制

• 批准号: 2141105
• 负责人: Terry Rogers Bishop
• 金额: $ 19.04万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-30 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2141105
• 关键词: antisense nucleic acid; apoptosis; complementary DNA; cytokine; erythroid stem cell; erythropoiesis; erythropoietin; gene expression; genetic library; growth factor receptors; high performance liquid chromatography; insulinlike growth factor; laboratory mouse; messenger RNA; northern blottings; nucleic acid sequence; oligonucleotides; polymerase chain reaction; protein kinase C; tissue /cell culture

We propose to elucidate mechanisms underlying maturation of erythroid colony-forming units (CFU-E) into red cells. Pursuing this goal, we use two populations of highly purified murine CFU-E: Generation I CFU-E forming 32-cell colonies, generation II forming 16-cell ones. For a rationale to dissect maturation, we draw upon our recent reports that (i) generation I CFU-E don't need erythropoietin (Epo) or other cytokines to progress to begins in generation II; (ii) DNA-breakdown characteristic of programmed cell death (apoptosis) first begins in generation II; (iii) Epo and insulin-like growth factor I (IGF-1) can then each almost completely forestall apoptosis; (iv) IGF-1 soon loses effectiveness whereas Epo remains effective until mid-maturation; (v) evident persistence of Epo action after it is withdrawn suggests that enduring transduction elements may have been unregulated by Epo; and (vi) for Epo- driven maturation to be optimal, a serum factor(s) other than IGF-1 is needed, especially when Epo levels are normal and this factor(s) then adds 30% or more to erythropoiesis. To identify factors (s) needed for optimal erythropoiesis, we apply reported candidates and serum fractions to well-defined serum-poor cultures to seek growth factors which can substitute for serum. We also seeks to learn why IGF-1 prematurely loses effectiveness and ask: It is due to loss of high-affinity receptors? In a parallel study, we better defined the time in CFU-E maturation when full-length Epo receptors (EpoR) capable of transducing prevention of apoptosis replace truncated EpoR which cannot. Results of such receptor analyses figure in our main purpose which is to identify molecular mechanisms by which apoptosis and cytokine response are transduced in CFU-E. Here we draw upon (i) genes whose expression in other tissues is associated with induction or prevention of apoptosis and (ii) gene expressed in CFU-E, especially those implicated by others in maturation and most especially those whose expression changes during maturation. Having established in preliminary studies that protein kinases (PK), particularly PKC, act in the pathway by which Epo forestalls CFU-E apoptosis, we focus on candidate PK whose changing transcription might underlie the temporal changes in apoptosis and/or cytokine response which we are observed. Drawing next upon pilot experiments which show that diverse PK clones can be isolated in varying recurrence frequencies from CFU-E RNA following reverse transcriptase polymerase-chain reactions primed with PK degenerate sequence oligomers, we plan to identify candidates PK stochastically. For example, using random PK clones developed from generation I CFU-E, from generation II plus Epo, from generation II plus IGF-1 and from generation II minus-cytokines, PK which recur unequally in the four CFU-E populations become candidates. Such PK, like non-PK candidates otherwise identified, will then be examined for maturation-dependent changes in RNA expression and, ultimately, for the influence their antisense oligomers have upon apoptosis and/or cytokine response. In these and other ways, we anticipate that elements underlying CFU-E maturation and apoptosis can be unabigously identified.

我们建议阐明红系成熟的机制 集落形成单位（CFU-E）进入红细胞。 为了实现这一目标，我们使用 两个高度纯化的鼠CFU-E群体：第I代CFU-E 形成32细胞集落，第二代形成16细胞集落。 用于 根据我们最近的报告，（i） 第一代CFU-E不需要促红细胞生成素（Epo）或其他细胞因子来 进展到第二代开始;（ii）DNA分解特征 程序性细胞死亡（凋亡）首先开始于第二代;（iii） Epo和胰岛素样生长因子I（IGF-1）几乎可以 完全阻止细胞凋亡;（iv）IGF-1很快失去效力 而Epo在成熟中期之前仍然有效;（v）明显 Epo作用在撤回后的持续性表明， 转导元件可能不受Epo调节;和（vi）对于Epo-转导元件， 驱动成熟是最佳的，除了IGF-1之外的血清因子是 需要，特别是当Epo水平正常时， 增加30%或更多的红细胞生成。 确定以下方面所需的因素： 最佳红细胞生成，我们应用报告的候选人和血清组分 到明确的血清缺乏培养，以寻找生长因子， 代替血清。 我们还试图了解为什么IGF-1过早失去 效力和问：这是由于高亲和力受体的损失？ 在 一项平行研究，我们更好地定义了CFU-E成熟的时间， 全长Epo受体（EpoR）能够转导预防 细胞凋亡取代不能的截短的EpoR。 这种受体的结果 我们的主要目的是分析图中的分子 细胞凋亡和细胞因子反应的机制被转导， CFU-E 在这里，我们利用（i）在其他组织中表达的基因， 与诱导或预防细胞凋亡相关的基因 在CFU-E中表达，特别是那些与成熟有关的 尤其是在成熟过程中表达发生变化的那些。 在初步研究中已经确定蛋白激酶（PK）， 尤其是PKC，在Epo阻止CFU-E的途径中起作用 细胞凋亡，我们专注于候选PK，其转录的变化可能 是细胞凋亡和/或细胞因子应答的时间变化的基础， 我们被观察。 接下来，根据试点实验， 不同的PK克隆可以以不同的复发频率从 逆转录聚合酶链反应后CFU-E RNA 用PK简并序列寡聚体引发，我们计划鉴定 候选人PK随机。 例如，使用随机PK克隆 从第一代CFU-E、第二代+Epo、 第二代加IGF-1和第二代减细胞因子， 在四个CFU-E群体中不均等地复发，成为候选者。 等 PK，像非PK候选人，以其他方式确定，然后将审查 对于RNA表达的成熟依赖性变化， 它们的反义寡聚体对细胞凋亡的影响和/或 细胞因子应答 通过这些和其他方式，我们预计， 潜在的CFU-E成熟和凋亡可以被清楚地鉴定。