Hydroxyurea Generates Nitric Oxide in Human Erythroid Cells: Mechanisms for γ-Globin Gene Activation

Abstract

Hydroxyurea (HU) induces fetal hemoglobin synthesis through activation of cyclic guanine monophosphate (cGMP) signaling. Studies in sickle cell patients demonstrated increased circulating nitric oxide (NO) levels after oral HU treatment. However, the direct measurement of NO in erythroid cells and its role in fetal hemoglobin induction have not been defined. Therefore, we quantified the level of nitrate and nitrite (NOx) generated by HU in human erythroid progenitors in the presence of three nitric oxide synthase inhibitors (NOS), including NG-monomethyl-L-arginine (L-NMMA). In addition, cGMP levels were measured in the presence or absence of the pathway inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, which blocks soluble guanylyl cyclase formation. HU treatment increased NOx levels and γ-globin transcription in K562 and primary erythroid cells, which was augmented when HU was combined with L-NMMA. Pretreatment with the cGMP pathway inhibitor reversed γ-gene activation by HU. These data demonstrate the direct stimulation of cellular NO and cGMP signaling in erythroid progenitors by HU as a possible mechanism for γ-globin gene activation.

Impact of hydroxyurea on clinical events in the BABY HUG trial

“…23 Unexpectedly, gastroenteritis was significantly less frequent in children taking hydroxyurea; this effect has not been previously reported in other adult or pediatric hydroxyurea studies. It is tempting to speculate that hydroxyurea's ability to replenish nitric oxide (NO) 24,25 may be related to this finding. NO improves gut motility 26 ; however, as described for priapism, 27 chronic NO depletion may change the sensitivity of smooth muscle to NO and disrupt normal protective feedback mechanisms, resulting in a robust response to a modest increase in NO because of infection/ inflammation.…”

Hydroxyurea responsiveness in -thalassemic patients is determined by the stress response adaptation of erythroid progenitors and their differentiation propensity

“…62 Although NO has been implicated in upregulation of HbF through activation of γ-globin expression, 63 NO also inhibits growth of erythroid primary cells and colony cultures. 64 Arginase hydrolyzes L-arginine to urea and L-ornithine in the urea NR5 NR3 NR2 NR1 NR8 NR6 NR4 R4 R7 R3 R1 R6 R2 R5 R8 NR7 cycle and inhibits nitric oxide (NO) production via competition with NOS for the substrate L-arginine.…”

Cytotoxicity and DNA damage in the neutrophils of patients with sickle cell anaemia treated with hydroxyurea

“…HU inhibits the enzyme ribonucleotide reductase (RNR), causes cell-cycle arrest, and allows globin genes to be more actively expressed. By killing cycling cells, HU changes the kinetics of erythroid proliferation, forcing more F cells to be produced from primitive progenitors and directly stimulating HbF production (Franco et al, 2006).Furthermore, HU therapy increases haemoglobin concentration, reduces the expression of adhesion molecules on erythrocytes, platelets and neutrophils, decreases the production of granulocytes and contributes to the improvement of clinical events, reducing the number of hospital admissions, the frequency of painful episodes, the need for transfusion and the occurrence of CVA and ACS (Platt et al, 1984;Steinberg et al, 2003;Zago, Pinto, 2007;Cartron, Elion, 2008;Orah, Platt, 2008;Conran, Franco-Penteado, Costa, 2009;Lou et al, 2009).…”