Nitric Oxide Synthase Dependency in Hydroxyurea Inhibition of Erythroid Progenitor Growth.

Tijana Subotički,Dragoslava Đikić,Juan F Santibanez,Milica Tošić,Vladan P Čokić,Olivera Mitrović Ajtić

doi:10.3390/genes12081145

Abstract

Hydroxyurea (HU) causes nitric oxide (NO) bioactivation, acting as both a NO donor and a stimulator of NO synthase (NOS). To examine whether HU effects are NO mediated by chemical degradation or enzymatic induction, we studied human and mouse erythroid cells during proliferation, apoptosis, and differentiation. The HU and NO donor demonstrated persisted versus temporary inhibition of erythroid cell growth during differentiation, as observed by γ- and β-globin gene expression. HU decreased the percentage of erythroleukemic K562 cells in the G2/M phase that was reversed by N-nitro l-arginine methyl ester hydrochloride (L-NAME). Besides activation of endothelial NOS, HU significantly increased apoptosis of K562 cells, again demonstrating NOS dependence. Administration of HU to mice significantly inhibited colony-forming unit-erythroid (CFU-E), mediated by NOS. Moreover, burst-forming-units-erythroid (BFU-E) and CFU-E ex vivo growth was inhibited by the administration of nitrate or nitrite to mice. Chronic in vivo NOS inhibition with L-NAME protected the bone marrow cellularity despite HU treatment of mice. NO metabolites and HU reduced the frequency of NOS-positive cells from CFU-E and BFU-E colonies that was reverted by NOS inhibition. HU regulation of the G2/M phase, apoptosis, differentiation, cellularity, and NOS immunoreactive cells was NOS dependent. Inhalation of NO therapy as well as strategies to increase endogenous NO production could replace or enhance HU activity.

Highlights

Transient nitric oxide (NO)-like radicals, from hydroxyurea, quench the tyrosyl free radical of the R2 subunit in ribonucleotide reductase [1]
In contrast to NO donors, the NO-releasing drug hydroxyurea demonstrated a continuous inhibition of erythroid cells growth, reaching 20–40% after day 6 (Figure 1C)
K562 cell growth was reduced with hydroxyurea after 24 h of incubation up to 40%, while prolonged treatment of 48 h showed more prominent inhibition up to 65%

Summary

Introduction

Transient nitric oxide (NO)-like radicals, from hydroxyurea, quench the tyrosyl free radical of the R2 subunit in ribonucleotide reductase [1]. The NO donors and inducible NO synthase (iNOS)-expressing cells reversibly inhibited DNA synthesis and erythroleukemic K562 cell growth due to the inhibitory interaction of NO with ribonucleotide reductase [6]. The percentage of S-phase and the total number of BFU-E were inversely correlated with fetal hemoglobin (HbF) levels in the peripheral blood of patients with sickle cell anemia treated with hydroxyurea, which reduced the total BFU-E colonies [10,11]. Hydroxyurea treatment increased total NO levels and γ-globin gene expression in K562 and primary erythroid cells, while NO donors augmented γ-globin and HbF expression in erythroid progenitors [12,13]. Hydroxyurea, in combination with the substrate l-arginine, increased NOS-dependent HbF synthesis in erythroid progenitors [14].

Results

Discussion

Conclusion