MOLECULAR CONTRIBUTORS TO STEM CELL QUIESCENCE

这项提议试图确定与调节干细胞静止有关的关键基因。干细胞、增殖祖细胞和有丝分裂后分化细胞之间平衡的破坏可能导致包括白血病和骨髓增殖性疾病在内的许多疾病状态。我们假设，p21和p27等细胞周期蛋白依赖性激酶抑制物(cdki‘s)在决定干细胞是处于静止状态还是开始循环时起着把关作用。我们已经证明，在静止的造血祖细胞(CD34 LIN和CD34 5-FU耐药)中，p21的表达升高。我们还证明了当静止的淋巴细胞被激活时，p27的亚细胞重新分布。我们认为，CDKI的人工下调将诱导静止的干细胞循环。这一建议将1.建立静止干细胞中p21、p16、p27和p57cdki的水平。这将使这些细胞周期抑制物被评估为维持干细胞处于静止状态的候选基因。这些蛋白质在候选干细胞中单独和协调的水平将被确定。由于尚未对干细胞中的细胞周期调节因子进行评估，通过这些实验获得的新信息将为解释干细胞行为建立遗传基准。2.检测细胞退出静止期后CDK1的亚细胞分布和表达的变化。我们预测，干细胞自我更新或产生后代细胞的承诺可能与CDKI细胞周期抑制蛋白水平的降低有关。为了确定哪些蛋白质变化诱导干细胞循环，首先必须确定当干细胞处于静止状态时，这些蛋白质的水平及其分布是如何变化的。3.确定CDKI下调是否足以将静止的干细胞招募到循环池中。要确定静止是否直接由候选CDKI的活动引起，需要功能上的确认。我们将开发和测试反义方法来调节干细胞中cdki的表达水平。最初的方法将是人为地下调p21，并测量这是否导致细胞处于静止状态。这些实验将利用细胞生物学中一个核心和长期存在的问题--干细胞的静止、自我更新和成熟之间的平衡--应用于细胞生长的分子控制方面的最新发现。从这些研究中获得的见解应该会对基因治疗策略产生重大影响，并应该为白血病和骨髓增生异常综合征的研究产生新的工具。

This proposal seeks to identify key genes involved in regulating stem cell quiescence. Disruption of the balance between stem cells, proliferating progenitor cells and post-mitotic differentiated cells can result in a number of disease states including leukemias and myeloproliferative disorders. We hypothesize that cyclin-dependent kinase inhibitors (cdki's) such as p2l and p27 serve as gatekeepers in determining whether stem cells remain quiescent or begin to cycle. We have demonstrated that the expression of p21 is elevated in quiescent hematopoietic progenitor (CD34+lin- and CD34+ 5-FU-resistant) cells. We also have demonstrated subcellular redistribution of p27 upon activation of quiescent lymphocytes. We propose that artificial downmodulation of cdki's will induce quiescent stem cells to cycle. This proposal will 1. Establish the levels of the p21, p16, p27 and p57 cdki's in quiescent stem cells. This will enable these cell cycle inhibitors to be evaluated as candidate genes for maintaining stem cells in the quiescent state. The levels of these proteins individually and coordinately in stem cell candidates will be determined. Because no evaluation of cell cycle regulators in stem cells has yet been undertaken, the novel information gained through these experiments will establish genetic benchmarks for interpreting stem cell behavior. 2. Determine the changes in the subcellular distribution and expression of cdki's as cells exit from quiescence. We predict that the commitment by stem cells to self-renew or to give rise to progeny cells is likely to be associated with a decrease in the levels of cdki cell-cycle inhibitor proteins. In order to determine which protein changes induce stem cells to cycle, one first must establish how levels of these proteins and their distribution change as stem cells leave quiescence. 3. Determine whether cdki downmodulation is sufficient for recruitment of quiescent stem cells into the cycling pool. Establishing whether quiescence directly results from the activity of candidate cdki's requires functional confirmation. We will develop and test anti-sense approaches to modulate cdki expression levels in stem cells. The initial approach will be artificially to downmodulate p21 and measure whether this causes cells to leave quiescence. These experiments will apply recent findings in the molecular controls on cell growth with a central and long-standing question in cell biology--the balance between quiescence, self-renewal and maturation of stem cells. Insights gained from these studies should have a significant impact on gene therapy strategies and should generate novel tools for the study of leukemias and myelodysplastic syndromes.