EFFECTS OF HYDROXYUREA ON FETAL HEMOGLOBIN SYNTHESIS BETA-GLOBIN DISORDERS

羟基脲对胎儿血红蛋白合成β-珠蛋白障碍的影响

• 批准号: 6289739
• 负责人: GRIFFIN P. RODGERS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289739
• 关键词: DNA footprinting; adult human (21+); blood disorder chemotherapy; clinical research; clinical trials; gene expression; gene induction /repression; globin; hemoglobin F; hemoprotein biosynthesis; human subject; human therapy evaluation; hydroxyurea; pharmacogenetics; pharmacokinetics; sickle cell anemia; thalassemia; tissue /cell culture; transcription factor

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

我们的团队专注于镰状细胞病和-地中海贫血患者胎儿血红蛋白水平的药理学增强。我们之前观察到羟基脲（HU）能够将约75-80%的镰状细胞病患者的胎儿血红蛋白（HbF）水平提高2-10倍，在成人患者的对照临床试验中达到顶峰，该试验明确证实了HU？S临床疗效。一项对52名连续参加HU试验的NIH患者的最新分析，包括最初的住院阶段和更长的门诊观察期，使我们能够得出关于反应率、对治疗反应的动力学方面和患者的最佳剂量计划的重要结论。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-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，我们发现，高浓度（即大于100微摩尔）的人成人红系细胞系统的HU处理伴随着HbF水平的2至4倍诱导，并且与γ球蛋白基因启动子近端区域三种特定转录因子或因子复合物的结合丧失有关。这一观察结果需要修改标准的体内足迹方法，称为ENU体内足迹，以允许识别延伸到G基元的足迹。随后的分析在一定程度上暗示了红系特异性转录因子GATA-1的表达受到抑制，以平行于这些HU浓度下红系细胞γ球蛋白表达和凋亡的增加。研究正在进行中，以确认这些观察结果的特异性，并进一步表征、纯化和最终克隆上游靶点和下游信号。我们还将这种液体红细胞培养方法与差分显示技术相结合，应用于检测在液体培养基系统中对人红细胞进行HU处理后诱导或抑制的新mRNA物种。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，并普遍表达；克隆52-12 85%同源于一个功能未知的高通量x连锁基因。我们相信这些基于实验室的研究可以阐明药物诱导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。-人体实验对象