Nutritional Cell Cycle and Differentiation Control

描述(申请人提供)：拟议的研究集中在维甲酸(RA)调节细胞生长和分化的复杂机制的一个方面。RA是饮食因子维生素A的活性代谢产物，维生素A是青少年正常发育所必需的；是胚胎发育过程中的形态生成物；以及调节肿瘤细胞生长和分化的化疗药物。这项研究利用了HL-60人类白血病细胞，这是一种未定向的造血前体细胞，可以经历髓系或单核细胞分化并停止生长。RA导致MAPK信号依赖的髓系分化和GO细胞停滞。这些研究将确定RA诱导的特定膜受体和适配器信号分子的表达对MAPK信号的意外激活和RA引起的基因表达变化的多样性的贡献。因此，他们关注于两个基本的生物学问题：1)MAPK信号在RA诱导的生长停滞和分化中的意义是什么，而这种信号通常被认为是有丝分裂的典型信号；2)RA引起的生长停滞和分化所需的不同顺序的基因表达变化是如何产生的。我们的实验室以及其他实验室使用差异显示、基于蛋白质组学的差异搜索或查询候选分子的组合搜索显示，RA诱导四种受体c-fms、BLR1、CD32和CD38的表达，这四种受体在特征上存在显著差异。因此，这些受体的下游信号结果可能不同，并调节不同基因集合的表达。然而，这些受体的一个共同特征是，它们都与利用MAPK信号有关。RA还可以诱导几个接头分子的表达，特别是Paxlin、SLP-76和dok。这些也与MAPK信号联系在一起，在某些情况下还明确地与上述一些受体有关。工作假说是RA诱导细胞表面受体的表达，其信号转导受适配分子的调节，导致MAPK信号的激活和相应的蛋白表达的变化，最终导致GO的停滞和分化。然后，受体和适配器的不同组合会直接导致不同蛋白质集合的表达变化，每种组合都会导致类风湿关节炎的总变化。本实验的目的是异位表达这些受体和适配器，无论是单独的还是联合的，以确定它们刺激MAPK信号转导和引起基因表达变化的能力，这些变化有助于RA诱导的整体变化，并测试它们的诱导表达在RA诱导的分化或GO抑制中的功能意义。

DESCRIPTION (provided by applicant): The proposed studies focus on one aspect of the complex mechanism by which retinoic acid (RA) regulates cell growth and differentiation. RA is the active metabolite of a dietary factor, vitamin A, necessary for proper development in juveniles; a morphogen during embryogenesis; and a chemotherapeutic agent regulating growth and differentiation of neoplastic cells. The studies utilize HL-60 human leukemia cells, an uncommitted hematopoietic precursor cell that can undergo myeloid or monocytic differentiation and GO arrest. RA causes MAPK signaling dependent myeloid differentiation and GO arrest of these cells. The studies will determine the contribution of RA-induced expression of selected membrane receptors and adapter signaling molecules to the unanticipated activation of MAPK signaling and to the diversity of gene expression changes caused by RA. They thus focus on two basic biological questions: 1) what is the significance of MAPK signaling in Ra-induced growth arrest and differentiation when such signaling is usually considered the prototypical signal for mitogenesis; 2) how are the diverse sequential gene expression changes needed to evoke growth arrest and differentiation generated by RA. Searches by our laboratory, as well as others, using a combination of differential display, a proteomics based differential search, or querying candidate molecules revealed that RA induces the expression of four receptors, c-FMS, BLR1, CD32, and CD38, which differ significantly in character. The downstream signaling consequences of these receptors are thus likely to differ and regulate the expression of different ensembles of genes. However, a common feature of these receptors is that all have been implicated with utilizing MAPK signaling. RA also induces the expression of several adapter molecules, in particular paxillin, SLP-76, and dok. These, too, have been connected with MAPK signaling and in some cases explicitly to some of the aforementioned receptors. The working hypothesis is that RA induces the expression of cell surface receptors, whose signaling is regulated by adapter molecules, resulting in MAPK signal activation and consequential changes in protein expression ultimately leading to GO arrest and differentiation. Different combinations of receptors and adapters then direct changes in the expression of different ensembles of proteins, each combination contributing to the total changes attributed to RA. The experimental aims are to ectopically express these receptors and adapters, singly and in combination, to determine their ability to stimulate MAPK signaling and cause gene expression changes that contribute to the total RA-induced changes, and to test the functional significance of their induced expression to RA-induced differentiation or GO arrest.