P-21 INDUCTION IN MYELOID DIFFERENTIATION

P-21 骨髓分化诱导

• 批准号: 2415658
• 负责人: RICHARD A STEINMAN
• 金额: $ 7.09万
• 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 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415658
• 关键词: CD antigens; CD34 molecule; Retroviridae; antisense nucleic acid; cell cycle; cell differentiation; cell growth regulation; flow cytometry; gene expression; gene induction /repression; guanine nucleotide binding protein; hematopoiesis; human tissue; mutant; myeloid stem cell; oncogenes; protein structure function; tissue /cell culture; transfection /expression vector

DESCRIPTION: (Adapted from Applicant's Abstract) The Applicant's central question is: In hematopoiesis what molecular events control the transition from cycling myeloid precursor cells to noncycling terminally differentiated cells? The PI hypothesizes that the cyclin dependent kinase inhibitor p21(WAFl CIPI) is responsible for this transition. The p21 prevents cells from progressing into S phase of the cell cycle, and mediates G1 arrest in response to p53 activation or other environmental cues. Numerous studies have confirmed the association between differentiation commitment and arrest in Gl phase of the cell cycle, but mechanisms of the linkage are incompletely understood. Based on the PI's observation that p21 is induced in immediate early fashion by myeloid differentiation inducers in cell lines, the PI postulates that p21 mediates cell cycle exit during myeloid differentiation. Specific aims of this proposal are: 1. To determine the stage specific expression of p21 in myelopoiesis and define the relationship of p21 expression to cell cycle stage and to the presence of CD11b, CD14, and CD15 antigen expression. This set of experiments will determine the normal variability of p21 levels as CFU GM cells differentiate and leave the mitotic compartment. Two channel FACScan analysis relating p21 expression to differentiation markers and to cell cycle status will be performed, and p21 message and protein will be measured in CD34+ cells under conditions promoting temporally coordinated granulocytic differentiation. 2. To uncover the effects of negative modulation of p21 activity on initiation and completion of the differentiation process. These experiments will address the questions: Is p21 expression required for the induction and maintenance of terminally differentiated cells? Antisense reagents and p21 mutants capable of abolishing endogenous p21 activity will be generated. The results of p21 downmodulation on myelopoiesis will be investigated first in HL60 cells. Experiments will then establish the effects of suppression of p21 on normal myeloid differentiation of CD34+ precursor cells in colony forming assays. Functional effects of p21 suppression on postmitotic differentiation also will be determined. 3. To determine whether forced expression of p21 enhances differentiation of murine and human myeloid progenitor cells; and to assess genetic and functional effects of forced expression of p21 in oncogene transformed myeloid progenitor cells exhibiting differentiation blockade. The p21 gene will be expressed aberrantly in myeloid cell lines using constitutive and inducible expression vectors, and in differentiating CD34+ cells using the MFG retroviral vector. Colony assays and FACScan analysis of differentiation markers will determine whether aberrant expression of p21 arrests cycling progenitor cells, accelerates differentiation, or otherwise disrupts normal myelopoiesis. Finally, the ability of p21 to suppress the growth and/or to permit differentiation of oncogene transformed cells will be determined. A clear understanding of p21 function in normal myelopoiesis will establish a context for determining if disruption of p21 function contributes to myelodysplastic syndrome or myeloid leukemias.

描述：（改编自申请人的摘要） 中心问题是：在造血中，哪些分子事件控制 从循环髓样前体细胞到非循环的过渡 终端分化的细胞？ PI假设细胞周期蛋白 依赖激酶抑制剂P21（WAFL CIPI）为此负责 过渡。 p21阻止细胞发展为S相 细胞周期，并介导G1停滞以响应P53激活 或其他环境提示。 许多研究证实了 差异化承诺与在GL阶段停止之间的关联 细胞周期的范围，但是连锁的机制是不完全的 理解。 根据PI的观察，P21在 细胞中的髓样分化诱导剂的立即早期时尚 线条，PI假设P21介导细胞周期出口 髓样分化。 该提议的具体目的是：1。 确定骨髓病中p21的阶段特异性表达 并定义p21表达与细胞周期阶段的关系 以及CD11b，CD14和CD15抗原表达的存在。 这组实验将确定 p21水平作为CFU GM细胞分化并留下有丝分裂 车厢。 两个通道FACSCAN分析与P21有关 对分化标记和细胞周期状态的表达将 进行，P21消息和蛋白质将在CD34+中进行测量 在促进时间协调的粒细胞的条件下的细胞 分化。 2。揭示负面调制的影响 p21启动和完成分化的活动 过程。 这些实验将解决以下问题：是P21 终端诱导和维护所需的表达 分化的细胞？ 反义试剂和P21突变体 将产生废除的内源性P21活性。 结果 将首先研究骨髓骨气的P21下调 在HL60细胞中。 然后，实验将确定 抑制P21在CD34+正常髓样分化上的抑制 菌落形成测定中的前体细胞。 P21的功能效应 还将确定抑制有丝分裂后分化。 3。确定强制表达p21是否增强 鼠和人髓样祖细胞的分化； 然后 评估p21强迫表达的遗传和功能效应 在癌基因转化的髓样祖细胞中 分化封锁。 p21基因将异常表达 使用本构和诱导表达向量的髓样细胞系， 并使用MFG逆转录病毒载体区分CD34+细胞。 分化标记的殖民分析和FACSCAN分析将 确定p21的异常表达是否阻止循环 祖细胞，加速分化或以其他方式破坏 正常的骨髓卵巢。 最后，p21抑制的能力 生长和/或允许分化癌基因转化的细胞 将确定。 对正常的P21功能有清晰的了解 骨髓卵子将建立一个环境来确定是否破坏 P21功能有助于骨髓增生综合征或髓样 白血病。