Pharmacological Control Of Human Hemoglobin Gene Expression

人类珠蛋白基因的个体发生是一个重要的研究焦点，无论是在基础发育生物学方面，还是在控制这一复杂基因系统表达的分子遗传学方面，而且还因为修改这种发育控制将在治疗常见的血红蛋白遗传疾病，如镰状细胞性贫血和地中海贫血中具有治疗价值。我们正在从几个方面研究这个问题。首先，我们改进了人类红细胞前体的培养方法，通过抗体选择，从而获得这些细胞的纯化群体，以建立在发育阶段药物改变其表型的方法。其次，我们开发了实时、定量的PCR方法来测量珠蛋白基因在单细胞中的表达，并利用这些方法分析了化学上有用的药物（如羟基脲和丁酸盐）对珠蛋白基因表达的影响机制。第三，我们已经证明，一氧化氮供体，如半胱氨酸- no，可以诱导K562细胞以及上述纯化的红细胞（CD34）前体细胞中的胎儿血红蛋白表达。通过γ -珠蛋白mRNA的定量PCR和胎儿血红蛋白的高效液相色谱法证实了胎儿血红蛋白的诱导作用。此外，我们已经证明no供体和羟基脲都通过刺激胍基环化酶和增加环GMP水平而起作用；相反，胍基环化酶抑制剂阻断了这两种药物的作用。几个实验室正在开发通过这种机制增加胎儿血红蛋白的药物，并准备进行临床试验。在最近的研究中，我们已经表明羟基脲诱导巨噬细胞和内皮细胞产生NO——部分是通过抑制eNOS蛋白的蛋白酶体降解——并且这种作用可能允许通过基质细胞和其他骨髓细胞产生的这些信号分子对红细胞祖细胞产生NO的旁分泌作用机制。在目前的研究中，在mRNA和蛋白质水平上，我们正在研究血红蛋白个体发生中这种信号传导的机制及其治疗意义。

The ontogeny of the human globin genes is an important focus of study both with respect to the fundamental developmental biology and molecular genetics of the control of expression of this complex gene system, but also because modifying this developmental control would be of therapeutic value in the treatment of the prevalent genetic diseases of hemoglobin, such as sickle cell anemia and thalassemia. We are studying this problem from several aspects. First, we have improved culture methods for human erythroid precursors, by antibody selection, so as to obtain purified populations of these cells to establish at which developmental stage drugs change their phenotype. Second, we have developed real-time, quantitative PCR methods to measure globin gene expression in single cells and have used these methods to analyze the mechanisms of the effects of chemically useful drugs, such as hydroxyurea and butyrate, on globin gene expression. Third, we have shown that nitric oxide donors, such as cysteine-NO, can induce fetal hemoglobin expression in K562 cells as well as the purified erythroid (CD34) precursor cells described above. Induction of fetal hemoglobin was demonstrated both by quantitative PCR of gamma-globin mRNA and by HPLC of fetal hemoglobin protein. Further, we have shown that both NO-donors and hydroxyurea acts by stimulating guanylyl cyclase and increasing cyclic GMP levels; conversely, inhibitors of guanylyl cyclase block the action of both agents. Agents to increase fetal hemoglobin by this mechanism are being developed in several laboratories and being readied for clinical testing. In more recent studies we have shown that hydroxyurea induces NO production in macrophages and endothelial cells-in part through inhibition of proteasome degradation of the eNOS protein-and that this efect may allow a mechanism for a paracrine efect of NO on erythroid progenitors from these signalling molecules produceed by stromal and other bone marrow cells. In current studies of globin production-at the mRNA and protein levels-we are studying the mechansims of such singaling in hemoglobin ontogeny and its therapeutic implications. Hydroxyurea, a drug widely used for treating myeloproliferative diseases, has also been approved for the treatment of sickle cell disease by raising fetal hemoglobin (HbF). We demonstrate now that during erythroid differentiation, endothelial NO synthase mRNA and protein levels decline steadily, as does the production of NO derivatives and cAMP levels, but cGMP levels are stable. Hydroxyurea increased intracellular cGMP levels and cAMP levels in erythroid progenitor cells (EPC). The NO donor, DEANONOate, induced much higher cGMP levels, but reduced cAMP levels. Hydroxyurea (1 mM) induced production of approximately 45 fmol cGMP/min/ng of purified sGC, similar to induction by 1 ?M of DEANONOate. We found that hydroxyurea and ProliNONOate produced iron-nitrosyl derivatives of sGC. Thus, we confirm that hydroxyurea can directly interact with the deoxy-heme of sGC, presumably by a free-radical nitroxide pathway, and activate cGMP production. These data add to an expanding appreciation of the role of hydroxyurea as an inducer of the NO/cGMP pathway in EPC. These mechanisms may also be involved in the cytostatic effects of hydroxyurea, as well as the induction of HbF. Overall, these results raise the possiblity that agents which affect this cyclic nucleotide pathway, or other signaling pathways, can be used to increase fetal hemoglobin levels in patients with sickle cell anemia and thalassemia. Such therapeutic advances have been the focus of this research program for almost three decades. In other recent studies we have been analyzing the change in fetal hemoglobin levels in normal newborns with age and find-using both quantitative protein and mRNA measurements-that there appear to be two distinct silencing mechansims for fetal hemoglobin-one primarily cellular and one primarily with respect to transcription mechanisms. We are now studying these processes in sickle cell children and will examine the effects of drugs on these silencing mechanisms. Further analyses of erythroid cells in culture are underway with respect to gene expression patterns in these cells and several manuscripts on this work have recently been submitted or published.