Hydroxyurea-Inducible Gene: Cloning and Characterization

已证明，羟基脲（HU）可增加胎儿血红蛋白（HbF）的生成，因此，它被用于治疗镰状细胞性贫血。虽然已经假设HU通过干扰红系前体的成熟间接促进HbF产生，但是不知道羟基脲如何调节g-珠蛋白表达的分子机制。为了阐明HU治疗后诱导HbF的潜在遗传途径，我们使用两相液体红细胞培养系统结合mRNA差异显示鉴定了HU诱导的基因。在阶段II期间的第6天和第7天用/不用100 μ M HU处理细胞，然后在第10天收集。通过凝胶电泳分析RT-PCR扩增的DNA片段，并进行HU处理或未处理的RNA样品的基因表达谱之间的比较。用56对随机引物在HU处理的样品中鉴定出383条特异性DNA带。经反向斑点杂交证实后，对8个表现出5倍以上基因诱导模式的阳性克隆进行测序，并在Genebank中搜索同源性。8个克隆中有一个与小鼠小GTP结合蛋白（sGBP）同源性为91%。我们进一步用3'和5' RACE从人骨髓cDNA文库中克隆了全长2.9Kb的cDNA序列。体外转录/翻译结果表明，该基因编码的多肽分子量为22 kDa，对应于一个594 bp的开放阅读框。这种小的GTP结合蛋白定位于6号染色体（带18.27），并在大多数组织中表达，特别是在甲状腺，脊髓和肾上腺中。为了确定其功能定位，我们将在3'端标记有GFP融合蛋白的该基因转染到K562和COS 7细胞中;我们发现该基因主要在Golgi-ER复合物中表达。为了确定该基因是否参与HU诱导g-珠蛋白基因表达，培养用该基因（或载体对照）稳定转染的K562细胞，并通过北方印迹测定g-珠蛋白基因的表达。与亲本K562细胞相比，sGBP-transfectants具有2.4倍的g-珠蛋白基因表达增加，而载体-transfectants仅表现出40%的g-珠蛋白诱导（P<0.05）。这些初步的数据表明，这种新的sGBP可能发挥作用的g-珠蛋白基因的诱导HU，和可能的信号转导途径的阐明目前正在调查中。

Hydroxyurea (HU) has been shown to augment the production of fetal hemoglobin (HbF) and for this reason is being used in the treatment of sickle cell anemia. While it has been assumed that HU promote HbF production indirectly by perturbing the maturation of erythroid precursors, the molecular mechanism(s) of how hydroxyurea regulates g-globin expression is not known. To illuminate potential genetic pathways responsible for the induction of HbF after HU treatment, we identified genes induced by HU using a two-phase liquid erythroid culture system in conjunction with mRNA differential display. Cells were treated with/without 100uM HU on days 6 and 7 during phase II, and then collected on day 10. RT-PCR-amplified DNA fragments were analyzed by gel electrophoresis, and a comparison between gene expression profiles of HU-treated or untreated RNA samples was performed. 383 unique DNA bands were identified in HU-treated samples using 56 sets of arbitrary primers. After confirmation by reverse dot blots, 8 positive clones exhibiting more than a 5-fold higher gene induction pattern were sequenced, and searched for homology in Genebank. One of 8 clones was found to be 91% homologous to a mouse small GTP binding protein (sGBP). We further cloned the full-length 2.9 Kb cDNA sequence from a human bone marrow cDNA library using 3' and 5' RACE. In vitro transcription/translation revealed a translated peptide of 22 kDa, corresponding to an open reading frame of 594bp. This small GTP-binding protein was localized to chromosome 6 (band 18.27) and is expressed in most tissues, especially in thyroid, spinal cord and adrenal gland. In order to define its functional localization, we transfected this gene tagged with a GFP fusion protein at the 3' end into K562 and COS7 cells; we found this gene expressed primarily in the Golgi-ER complex. In order to determine whether this gene was involved in the induction of the g-globin gene expression by HU, K562 cells stably transfected with this gene (or vector control) were grown and the expression of g-globin gene was determined by Northern Blots. sGBP-transfectants had a 2.4 fold increase in g-globin gene expression, whereas, vector-transfectants only demonstrated a 40% g-globin induction, both compared with parental K562 cells (P<0.05). These preliminary data suggest that this novel sGBP may play a role in the induction of g-globin gene by HU, and an elucidation of possible signal transduction pathways is currently under investigation.