EFFECTS OF HYDROXYUREA ON FETAL HEMOGLOBIN SYNTHESIS BETA-GLOBIN DISORDERS

我们的研究小组主要关注镰状细胞病和β地中海贫血患者胎儿血红蛋白水平的药物增加。我们以前的观察表明，羟基脲（HU）能够增加胎儿血红蛋白（HbF）水平在约75-80%的镰状细胞病患者的2-10倍，最终在对照临床试验中的成年患者，证实明确HU？的临床疗效。对进入HU试验的52名连续NIH患者进行了更新分析，包括最初的住院期和更长的门诊观察期，这使我们能够得出关于反应率，对治疗反应的动力学方面以及患者的最佳剂量方案的重要结论。此外，在中国和泰国正在进行的合作研究中，我们发现一些不依赖输血的中间型β地中海贫血患者可能主要通过改善新形成的RBC的质量而受益于HU治疗，从而增加他们的血细胞计数。在相关的实验室研究中，我们已经发现，在高浓度（即大于100微摩尔）的人成人红系细胞系统下的HU处理伴随着HbF水平的2至4倍诱导，并且与γ珠蛋白基因启动子的近端区域中的三种特异性转录因子或因子复合物的结合丧失相关。这一观察结果需要修改标准体内足迹法，称为ENU体内足迹法，以允许识别延伸到 G 图案随后的分析部分地暗示了红系特异性转录因子加塔-1表达的抑制，与在这些HU浓度下增加的γ珠蛋白表达和红系细胞凋亡平行。研究正在进行中，以确认这些观察结果的特异性，以及进一步表征，纯化，并最终克隆上游靶点和下游信号。我们还将这种液体红系细胞培养方法与差异显示技术相结合，应用于检测在液体培养基系统中HU处理人红系细胞后诱导或抑制的新mRNA种类。我们已经确定了两个有趣的候选基因？克隆39-13与小鼠ras-样GTP结合蛋白具有91%的同源性，并且广泛表达;克隆52-12与未知功能的高通量X-连锁基因具有85%的同源性。我们相信，这些基于实验室的研究可能会澄清药理学诱导的胎儿血红蛋白增加的分子机制，并可能揭示其他有前途的化合物。- 人类受试者

Our group has focused on the pharmacological augmentation of fetal hemoglobin levels in patients with sickle cell disease and beta- thalassemia. Our previous observations that hydroxyurea (HU) is capable of increasing fetal hemoglobin (HbF) levels in about 75-80% of patients with sickle cell disease by 2-10 fold, culminated in a controlled clinical trial in adult patients which substantiated unambiguously HU?s clinical efficacy. An updated analysis of 52 consecutive NIH patients entered on the HU trial, consisting of an initial in-hospital phase, and more prolonged outpatient observation periods has allowed us to draw important conclusions about the response rate, the kinetic aspects of the response to therapy, and the optimal dosage schedules for patients. In addition, in on-going collaborative studies in China and Thailand we have found that some patients with beta-thalassemia intermedia, who are not transfusion-dependent may benefit from HU treatment mainly by improving the quality of the newly formed RBC, and thereby increase their blood counts. In related laboratory studies, we have found that HU treatment at high concentrations (i.e. greater than 100 micro-molar) of a human adult erythroid cell system, is accompanied by a 2 to 4-fold induction of HbF levels and is associated with the loss of binding of three specific transcription factors or factor complexes in the proximal region of the gamma globin gene promoter. This observation required the modification of the standard in vivo footprinting methodology, termed ENU in vivo footprinting, to permit the identification of footprints extending into G motifs. Subsequent analysis has in part implicated an inhibition in the expression of the erythroid specific transcription factor, GATA-1, to parallel the increase gamma globin expression and apoptosis of erythroid cells at these HU concentrations. Studies are underway to confirm the specificity of these observations as well as to further characterize, purify, and eventually clone the upstream targets and downstream signals. We have also coupled this liquid erythroid culture methodology with the technique of differential display has been applied to examine for novel mRNA species induced or repressed following HU treatment of human erythroid cell in a liquid medium system. We have identified two interesting candidate genes ? clone 39-13 shares 91% homology with the mouse ras-like GTP binding protein and is ubiquitously expressed; clone 52-12 is 85% homologous to a high-throughput X-linked gene of unknown function. We believe that these lab-based studies may clarify the molecular mechanism of pharmacological-induced fetal hemoglobin increases, and may shed light on other promising compounds. - Human Subjects