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Hydroxyurea ( HU) is a drug used for the treatment of haemoglobinopathies. Hydroxyurea functions by upregulating γ-globin transcription and fetal haemoglobin ( Hb F) production in erythroid cells. The K562 erythroleukaemia cell line is widely used as a model system in which to study the mechanism of γ-globin induction by HU. However, the transcription factors required for the upregulation of γ-globin expression by HU in K562 cells have not been identified. Similarities between the HU and sodium butyrate ( SB) pathways suggest cAMP response element-binding protein ( CREB) 1 as a potential candidate. Thus, the aim of the present study was to investigate the possible role of CREB1 in the HU pathway., Experiments were performed using transient and stable RNA interference ( RNAi) to show that CREB1 is necessary for HU-mediated induction of γ-globin expression and haemoglobin production in K562 cells., Furthermore, western blot analyses demonstrated that CREB1 becomes phosphorylated in a dose-dependent manner after HU (100-400 µmol/ L) treatment of K562 cells for 72 h., We also investigated role of a Gγ promoter CREB1 response element ( G- CRE) in this pathway. Quantitative amplification refractory mutation system-polymerase chain reaction experiments were performed to demonstrate that HU induces the expression of both Gγ and Aγ in this cell line. In addition, electrophoretic mobility shift assays were used to show that levels of CREB1 complexes binding to the G- CRE site are increased following HU treatment and are decreased in CREB1-knockdown cells., The results suggest that CREB1 is necessary for γ-globin induction by HU in K562 cells, a role that may be mediated, in part, through the G- CRE element. [ABSTRACT FROM AUTHOR]

1. Hydroxyurea (HU) is a drug used for the treatment of haemoglobinopathies. Hydroxyurea functions by upregulating γ-globin transcription and fetal haemoglobin (HbF) production in erythroid cells. The K562 erythroleukaemia cell line is widely used as a model system in which to study the mechanism of γ-globin induction by HU. However, the transcription factors required for the upregulation of γ-globin expression by HU in K562 cells have not been identified. Similarities between the HU and sodium butyrate (SB) pathways suggest cAMP response element-binding protein (CREB) 1 as a potential candidate. Thus, the aim of the present study was to investigate the possible role of CREB1 in the HU pathway. 2. Experiments were performed using transient and stable RNA interference (RNAi) to show that CREB1 is necessary for HU-mediated induction of γ-globin expression and haemoglobin production in K562 cells. 3. Furthermore, western blot analyses demonstrated that CREB1 becomes phosphorylated in a dose-dependent manner after HU (100-400 µmol/L) treatment of K562 cells for 72 h. 4. We also investigated role of a Gγ promoter CREB1 response element (G-CRE) in this pathway. Quantitative amplification refractory mutation system-polymerase chain reaction experiments were performed to demonstrate that HU induces the expression of both Gγ and Aγ in this cell line. In addition, electrophoretic mobility shift assays were used to show that levels of CREB1 complexes binding to the G-CRE site are increased following HU treatment and are decreased in CREB1-knockdown cells. 5. The results suggest that CREB1 is necessary for γ-globin induction by HU in K562 cells, a role that may be mediated, in part, through the G-CRE element., (© 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Blackwell Publishing Asia Pty Ltd.)