Cloning/Characterization Of A Hydroxyurea-inducible Gene

羟基脲诱导基因的克隆/表征

• 批准号: 7151524
• 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/7151524
• 关键词: apoptosis; cell cycle; differential display technique; erythropoiesis; erythropoietin; gene expression; gene induction /repression; gene therapy; genetic regulation; guanine nucleotide binding protein; hemoglobin F; human tissue; hydroxyurea; immunoglobulin genes; polymerase chain reaction; protein structure function; protein transport; small interfering RNA; tissue /cell culture; transfection

Hydroxyurea (HU), a drug effective in the treatment of sickle cell disease, is thought to indirectly promote fetal hemoglobin (Hb F) production by perturbing the maturation of erythroid precursors. The molecular mechanisms involved in HU-mediated regulation of gamma-globin expression currently remain unclear. We identified a HU induced small GTP-binding protein, secretion-associated and ras-related (SAR) in adult erythroid cells by differential display. Stable SAR expression in K562 cells resulted in macrocytosis and immature cells appearance associated with increased gamma-globin mRNA. SAR-mediated induction of the gamma-globin gene also inhibited K562 cell growth including arrest in G1/S phase, apoptosis and delay of maturation, cellular changes consistent with the previously known effects of HU on erythroid cells. Similarly, SAR also enhanced both gamma-and beta-globin transcription in bone marrow primary CD34+ cells, but its effects on gamma-globin induction is more profound than that on beta-globin. Though upregulation of GATA-2 and p21 were observed both in SAR-expressing cells and HU-treated K562 cells, the PI3 kinase and phosphorylated ERK were inhibited specifically in SAR-expressing cells. These data reveal a novel role of SAR distinct from its previously known protein trafficking function. We suggest that SAR may participate in both erythroid cell growth and gamma-globin production by regulating PI3-kinase/ERK and GATA-2/p21 dependent signal transduction pathways. Further elucidation of the involved pathways utilizing siRNA and specific protein trafficking inhibitors, as well as an analysis of the function of retroviral delivered SAR into primary hematopoietic cell are currently underway. Deletion and mutation studies of the SAR promoter have also been initiated to define potential HU-response site(s).

羟基脲(HU)是一种治疗镰状细胞疾病的有效药物，被认为是通过干扰红系前体细胞的成熟而间接促进胎儿血红蛋白(Hb-F)的产生。目前，HU介导的伽玛珠蛋白表达调控的分子机制尚不清楚。通过差异显示，我们在成人红系细胞中发现了一种HU诱导的小GTP结合蛋白，分泌相关和ras相关(SAR)。K562细胞中稳定的SAR表达导致巨噬细胞增多和幼稚细胞的出现，并伴随着γ-珠蛋白mRNA的增加。SAR介导的γ-珠蛋白基因的诱导也抑制了K562细胞的生长，包括使K562细胞停滞在G1/S期，细胞凋亡和成熟延迟，细胞变化与已知的HU对红系细胞的作用一致。类似地，SAR也促进了骨髓原代CD34+细胞中的γ-珠蛋白和β-珠蛋白的转录，但其对γ-珠蛋白的诱导作用比对β-珠蛋白的影响更深远。虽然在表达SAR的细胞和经HU处理的K562细胞中都观察到GATA-2和p21的上调，但在表达SAR的细胞中，PI3激酶和磷酸化的ERK被特异性地抑制。这些数据揭示了SAR不同于先前已知的蛋白质运输功能的新作用。我们认为，SAR可能通过调节依赖于PI3-K/ERK和GATA-2/p21的信号转导途径，参与红系细胞的生长和丙种球蛋白的产生。利用siRNA和特定的蛋白转运抑制剂进一步阐明所涉及的途径，以及分析逆转录病毒携带的SAR进入原代造血细胞的功能目前正在进行中。此外，还启动了对SAR启动子的缺失和突变研究，以确定潜在的Hu-Response位点(S)。