MECHANISMS OF SYNERGISTIC REGULATION OF STEM/PROGENITORS

细胞因子在体外刺激、增强和/或抑制髓系祖细胞（MPC）的增殖。一些细胞因子在体外的作用已经在动物模型中重现，一些在人体临床试验中显示出疗效。体外观察到的协同作用很可能与体内造血有关，即细胞因子组合对MPC增殖的刺激，尤其是强效的共刺激细胞因子如钢铁因子，以及趋化因子对这种协同作用的抑制。我们相信，对这些促进和抑制生长的细胞因子引发的细胞内机制的评估将增强我们对正常MPC调节的理解，并最终用于为血液病患者设计合理的治疗方法。我们认为，多种生长因子诱导的MPC增殖的协同刺激和趋化因子的抑制，至少在一定程度上是细胞周期现象。我们的假设是细胞周期调节因子，如周期蛋白依赖性激酶抑制剂，p21cip1/waf1，某些周期蛋白依赖性激酶和周期蛋白，以及其他细胞内信号是介导增殖协同反应的关键。我们的生化结果和对功能缺失这些细胞内介质的小鼠MPC增殖的评估支持这一假设。我们的长期目标是确定参与这些作用的关键细胞内分子，并利用这些信息进行临床获益。为此，我们提出以下两个具体目标：探讨多种生长因子（GM-CSF和steel因子）诱导的MPC增殖协同刺激的细胞内机制：a)评估周期蛋白依赖性激酶抑制剂/调节剂p21cip1/waf1的潜在作用，及其与周期蛋白依赖性激酶和周期蛋白在这些作用中的关系；b)阐明转录因子BCL-6和Stat4、蛋白磷酸酶SHP-1和SHIP、MAP激酶的潜在作用，以及它们之间以及它们与p21cip1/waf1之间可能的相互关系。2. 通过评估骨髓抑郁性CC （mip -1 α、MCP-1）、CXC （IL-8、IP-10）和C（淋巴趋化因子）趋化因子及其作用于的受体的比较作用，评估参与抑制多种生长因子诱导的MPC增殖协同刺激的细胞内机制：a)细胞周期和控制细胞周期进展的调节分子；b)与MPC增殖刺激相关的细胞内信号分子。

Cytokines act in vitro to stimulate, enhance, and/or suppress proliferation of myeloid progenitor cells (MPC). Actions of some cytokines noted in vitro have been reproduced in animal models, and some have shown efficacy in human clinical trials. It is probable that synergism noted in vitro in terms of stimulation of MPC proliferation in response to combinations of cytokines, especially a potent co-stimulating cytokine such as steel factor, and suppression of this synergism by chemokines are of relevance to hematopoiesis in vivo. It is our belief that an evaluation of intracellular mechanisms triggered by these growth promoting and suppressing cytokines will enhance our understanding of normal MPC regulation, and will ultimately be of use in designing rational treatments for patients with hematological disorders. We believe that multi-growth factor induced synergistic stimulation of MPC proliferation and this inhibition by chemokines is, at least in part, a cell cycle phenomenon. Our hypothesis is that cell cycle regulators such as cyclin-dependent kinase inhibitor, p21cip1/waf1, certain cyclin-dependent kinases, and cyclins, and other intracellular signals are key to responses mediating proliferative synergy. Our biochemical results and evaluation of MPC proliferation from mice functionally deleted in some of these proposed intracellular mediators support this hypothesis. Our long-term goal is to define key intracellular molecules involved in these effects and to utilize this information for clinical benefit. Towards these goals we propose the following two Specific Aims: 1. Investigate intracellular mechanisms involved in multiple growth factor (GM-CSF and steel factor)-induced synergistic stimulation of the proliferation of MPC by: a) assessing a potential role for the cyclin-dependent kinase inhibitor/modulator p21cip1/waf1, and its relationship with cyclin-dependent kinases and cyclins in these effects and b) elucidating a potential role for the transcription factors BCL-6 and Stat4, the protein phosphatases SHP-1 and SHIP, and MAP kinase, and their possible interrelationship(s) with each other and with p21cip1/waf1 in these effects. 2. Evaluate intracellular mechanisms involved in suppression of multiple growth factor-induced synergistic stimulation of the proliferation of MPC by assessing comparative effects of myelosuppressive CC (MIP-1alpha, MCP-1), CXC (IL-8, IP-10), and C (lymphotactin) chemokines and the receptors they act on through: a) the cell cycle and regulatory molecules which control cell cycle progression and b) Intracellular signaling molecules linked to stimulation of MPC proliferation.