Effects Of Hydroxyurea On Fetal Hemoglobin Synthesis Bet

羟基脲对胎儿血红蛋白合成的影响

• 批准号: 7151520
• 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/7151520
• 关键词: apoptosis; blood disorder chemotherapy; cell cycle; cell line; cysteine endopeptidases; cytotoxicity; dosage; drug administration rate /duration; drug screening /evaluation; erythropoiesis; gene expression; gene induction /repression; globin; hemoglobin F; hemoprotein; hemoprotein biosynthesis; hemoprotein structure; human subject; human therapy evaluation; hydroxyurea; microarray technology; patient oriented research; pharmacogenetics; pharmacokinetics; polymerase chain reaction; sickle cell anemia; sickling inhibitor; thalassemia; tissue /cell culture; transcription factor

Hydroxyurea (HU) has been shown to increase the proportion of fetal hemoglobin (Hb F) in most sickle cell patients. A low dosage regimen of HU increased total hemoglobin (Hb) levels in some thalassemia intermedia patients by preferentially increasing b-globin biosynthesis. We examined the effect of dosage, duration of exposure, and developmental stage at which HU was given on various hematological parameters and signaling pathways. Erythroid cells in two-phase liquid culture were exposed to HU (5 to 100 mM) either as a pulse for 3 days or continuously for 12 days. Low doses of HU (from 0 to 25 mM) increased Hb levels by up to 2.7-fold, and a high dose of HU (100 mM) increased Hb levels when added at days 3-6 of phase II. No significant changes in Hb levels were observed in response to HU during the late stage of phase II culture (>=9-12 days), although there was a significant dose-dependent increase in Hb F levels. HU exposure during days 0-3 of phase II culture increased the number of erythroid colonies to a maximum of 5-fold at 5 mM HU. GATA-1 mRNA was down-regulated at a high dose of HU and GATA-2 was dose-dependently up-regulated over a lower dosage range. Treatment with 100 mM HU dramatically upregulated the death receptor DR-5, caspase 3, and various other genes related to cell cycle control and apoptosis, as determined by cDNA microarray analysis. In contrast, 10 mM HU modestly up-regulated mRNA levels of the early growth response gene (egr-1). More recently, we used the human erythroleukemia cell line (K562) cells to examine the molecular effects of HU at 4 doses: 0, 10, 100, and 1000 umol/l in 4-day cell culture. Our results confirmed that HU could induce the expression of human gamma-globin gene and there was a dose related decrease in cell growth when K562 cells were incubated with HU. From 8534 screened genes of cDNA microarray analyses with 4-dose-treated K562 cells with HU in two independent experiments, 6580 differentially expressed genes were found, among which 194 genes whose expression was modulated at least 2-fold in K562 cells. Taken together our results indicate that HU exerts concentration-dependent effects on Hb F production and erythropoiesis and that these two effects are mediated by distinct molecular mechanisms.

在大多数镰状细胞病患者中，已证明羟基脲（HU）可增加胎儿血红蛋白（Hb F）的比例。低剂量的HU方案通过优先增加b-珠蛋白生物合成来增加某些中间型地中海贫血患者的总血红蛋白（Hb）水平。我们研究了剂量的影响，暴露的持续时间，和发展阶段，在此HU给予各种血液学参数和信号通路。将两相液体培养物中的红系细胞暴露于HU（5至100 mM），脉冲3天或连续12天。低剂量的HU（0至25 mM）使Hb水平增加高达2.7倍，并且高剂量的HU（100 mM）当在阶段II的第3-6天添加时使Hb水平增加。在II期培养的后期阶段（>=9-12天），没有观察到响应HU的Hb水平的显著变化，尽管Hb F水平存在显著的剂量依赖性增加。在阶段II培养的第0-3天期间，HU暴露使红细胞集落的数量在5 mM HU下增加至最大5倍。加塔-1 mRNA在高剂量HU下下调，而加塔-2在较低剂量范围内呈剂量依赖性上调。用100 mM HU处理显著上调死亡受体DR-5、半胱天冬酶3和与细胞周期控制和凋亡相关的各种其他基因，如通过cDNA微阵列分析所确定的。相比之下，10 mM HU适度上调早期生长反应基因（ERF-1）的mRNA水平。最近，我们使用人红白血病细胞系（K562）细胞，以检查HU在4天细胞培养中以4种剂量（0、10、100和1000 umol/l）的分子效应。结果表明，HU能诱导人γ-珠蛋白基因的表达，并能抑制K562细胞的生长，且与剂量有关。在两个独立实验中，从8534个基因的cDNA微阵列分析中筛选出6580个差异表达基因，其中194个基因在K562细胞中的表达被调节至少2倍。两者合计，我们的研究结果表明，胡施加浓度依赖性的影响，血红蛋白F生产和红细胞生成，这两种效果是由不同的分子机制介导的。