Effects Of Hydroxyurea On Fetal Hemoglobin Synthesis Bet

在大多数镰状细胞患者中，羟基尿素(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-珠蛋白的生物合成，提高了一些中间地中海贫血患者的总血红蛋白(Hb)水平。我们研究了剂量、暴露时间和给予HU的发育阶段对各种血液学参数和信号通路的影响。将两相液体培养的红系细胞以脉冲形式暴露于HU(5~100 mm)3天或连续12天。低剂量的HU(从0到25 mM)使Hb水平增加2.7倍，高剂量的HU(100 MM)在II期培养的第3-6天增加Hb水平。在II期培养的后期(9-12天)，HU对Hb水平没有明显的影响，尽管Hb F水平有显著的剂量依赖性增加。在第二阶段培养的第0-3天，HU暴露使红系集落数在5 mMHU时达到最大值5倍。高剂量组GATA-1mRNA表达下调，低剂量组GATA-2表达呈剂量依赖性上调。通过基因芯片分析，100 mM HU处理显著上调死亡受体DR-5、caspase3和其他与细胞周期控制和细胞凋亡相关的基因。相反，10 mM HU可适度上调早期生长反应基因(EGR-1)的mRNA水平。最近，我们使用人红白血病细胞系(K562)细胞，在4天的细胞培养中检测了HU 4个剂量：0，10,100和1000umol/L的分子效应。我们的结果证实了HU可以诱导人γ-珠蛋白基因的表达，并且HU对K562细胞的生长有剂量依赖性的抑制作用。两次独立实验共筛选出8534个经HU处理的K562细胞的差异表达基因，发现6580个差异表达基因，其中194个基因在K562细胞中表达上调2倍以上。综上所述，我们的结果表明，HU对Hb-F的产生和红细胞生成具有浓度依赖性的影响，并且这两种作用是由不同的分子机制介导的。

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.