Hydroxyurea nitrosylates and activates soluble guanylyl cyclase in human erythroid cells

Laboratory of Experimental Hematology, Institute for Medical Research, Belgrade, Serbia.
Blood (Impact Factor: 10.45). 03/2008; 111(3):1117-23. DOI: 10.1182/blood-2007-05-088732
Source: PubMed


Hydroxyurea, a drug widely used for treating myeloproliferative diseases, has also been approved for the treatment of sickle cell disease by raising fetal hemoglobin (HbF). We have shown that nitric oxide (NO) and the soluble guanylyl cyclase (sGC) pathways are involved in hydroxyurea induction of HbF levels in erythroid progenitor cells (EPCs). We demonstrate now that during erythroid differentiation, endothelial NO synthase mRNA and protein levels decline steadily, as does the production of NO derivatives and cyclic adenosine monophosphate (cAMP) levels, but guanosine 3',5'-cyclic monophosphate (cGMP) levels are stable. Hydroxyurea increased intracellular cGMP levels and cAMP levels in EPCs. The NO donor, DEANONOate, induced much higher cGMP levels, but reduced cAMP levels. Hydroxyurea (1 mM) induced production of approximately 45 pM cGMP/minute/ng of purified sGC, similar to induction by 1 muM DEANONOate. We found that hydroxyurea and ProliNONOate produced iron-nitrosyl derivatives of sGC. Thus, we confirm that hydroxyurea can directly interact with the deoxy-heme of sGC, presumably by a free-radical nitroxide pathway, and activate cGMP production. These data add to an expanding appreciation of the role of hydroxyurea as an inducer of the NO/cGMP pathway in EPCs. These mechanisms may also be involved in the cytostatic effects of hydroxyurea, as well as the induction of HbF.

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Available from: Silvana Andric
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    • "Hb F induction by HU has been correlated to cell cycle inhibition leading to activation of stress erythropoiesis [1,19-21]. Other studies have suggested that Hb F induction by HU is mediated more specifically via nitric oxide-dependent transcriptional mechanisms [22,23] and cyclic nucleotides [24,25] and initial evidence for some epigenetic regulation [26]. "
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    ABSTRACT: Sickle cell anemia (SCA) presents a complex pathophysiology which can be affected by a number of modifying factors, including genetic and biochemical ones. In Brazil, there have been no studies verifying betaS-haplotypes effect on oxidative stress parameters. This study evaluated betaS-haplotypes and Hb F levels effects on oxidative stress markers and their relationship with hydroxyurea (HU) treatment in SCA patients. The studied group was composed by 28 SCA patients. Thirteen of these patients were treated with HU and 15 of them were not. We used molecular methodology (PCR-RFLP) for hemoglobin S genotype confirmation and haplotypes identification. Biochemical parameters were measured using spectrophotometric methods (Thiobarbituric-acid-reactive substances and Trolox equivalent antioxidant capacity levels, catalase and GST activities) and plasma glutathione levels by High-performance liquid chromatography coupled to electrochemical detection. We found the highest frequency of Bantu haplotype (48.2%) which was followed by Benin (32.1%). We observed also the presence of Cameroon haplotype, rare in Brazilian population and 19.7% of atypical haplotypes. The protective Hb F effect was confirmed in SCA patients because these patients showed an increase in Hb F levels that resulted in a 41.3% decrease on the lipid peroxidation levels (r =-0.74, p=0.01). Other biochemical parameters have not shown differential expression according to patient's haplotypes. Bantu haplotype presence was related to the highest lipid peroxidation levels in patients (p < 0,01), but it also conferred a differential response to HU treatment, raising Hb F levels in 52.6% (p = 0.03) when compared with the group with the same molecular profile without HU usage. SCA patients with Bantu haplotype showed the worst oxidative status. however these patients also demonstrated a better response to the treatment with HU. Such treatment seems to have presented a "haplotype-dependent" pharmacological effect.
    Full-text · Article · Oct 2013 · BMC Medical Genetics
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    • "We did not find statistical significance in the percentage of CD34, CD36 and GlyA antigen positive cells among erythroid progenitors derived from examined hematopoietic cells (Figure 1B). Besides flow cytometry for analysis of in vitro erythroid differentiation, we already reported measurement of hemoglobin content by benzidine staining and high-performance liquid chromatography in erythroid progenitor cells during their in vitro differentiation in the same culture conditions [6,7]. "
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    ABSTRACT: Background The guanine nucleotide binding protein (G protein)-coupled receptors (GPCRs) regulate cell growth, proliferation and differentiation. G proteins are also implicated in erythroid differentiation, and some of them are expressed principally in hematopoietic cells. GPCRs-linked NO/cGMP and p38 MAPK signaling pathways already demonstrated potency for globin gene stimulation. By analyzing erythroid progenitors, derived from hematopoietic cells through in vitro ontogeny, our study intends to determine early markers and signaling pathways of globin gene regulation and their relation to GPCR expression. Results Human hematopoietic CD34+ progenitors are isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and G-CSF stimulated mobilized PB (mPB), and then differentiated in vitro into erythroid progenitors. We find that growth capacity is most abundant in FL- and CB-derived erythroid cells. The erythroid progenitor cells are sorted as 100% CD71+, but we did not find statistical significance in the variations of CD34, CD36 and GlyA antigens and that confirms similarity in maturation of studied ontogenic periods. During ontogeny, beta-globin gene expression reaches maximum levels in cells of adult blood origin (176 fmol/μg), while gamma-globin gene expression is consistently up-regulated in CB-derived cells (60 fmol/μg). During gamma-globin induction by hydroxycarbamide, we identify stimulated GPCRs (PTGDR, PTGER1) and GPCRs-coupled genes known to be activated via the cAMP/PKA (ADIPOQ), MAPK pathway (JUN) and NO/cGMP (PRPF18) signaling pathways. During ontogeny, GPR45 and ARRDC1 genes have the most prominent expression in FL-derived erythroid progenitor cells, GNL3 and GRP65 genes in CB-derived cells (high gamma-globin gene expression), GPR110 and GNG10 in BM-derived cells, GPR89C and GPR172A in PB-derived cells, and GPR44 and GNAQ genes in mPB-derived cells (high beta-globin gene expression). Conclusions These results demonstrate the concomitant activity of GPCR-coupled genes and related signaling pathways during erythropoietic stimulation of globin genes. In accordance with previous reports, the stimulation of GPCRs supports the postulated connection between cAMP/PKA and NO/cGMP pathways in activation of γ-globin expression, via JUN and p38 MAPK signaling.
    Full-text · Article · Feb 2013 · BMC Genomics
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    • ", [42] HbF MYB (Guanylyl cyclase, GC), cGMP [43] , [44] , cGMP MYB [14] [45] , "
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    ABSTRACT: Fetal hemoglobin (HbF) is the main type of hemoglobin in the fetus and few in adult, but retains high levels in some people and patients with beta-thalassemia major or sickle cell disease. High HbF levels are beneficial to ameliorating the disease severity of the anemia. Previous researches had established that quantitative trait loci were associated with 6q23 and 2p15. Recent researches indicated that HBS1L-MYB in 6q23 and BCL11A in 2p15 are highly correlated to HbF levels. These discoveries not only help to understanding of mechanism in HbF expression, but also provide potential drug targets for therapy of sickle cell disease. The progress on genes related to fetal hemoglobin quantitative trait and potential applications was summarized in this review.
    Preview · Article · Apr 2010 · Hereditas (Beijing)
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