Nitric Oxide Synthase Dependency in Hydroxyurea Inhibition of Erythroid Progenitor Growth

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.

Nitric Oxide Mediation in Hydroxyurea and Nitric Oxide Metabolites’ Inhibition of Erythroid Progenitor Growth

“…The experimental protocol was already described [ 5 ] and approved by the Ethics Committee of the Institute for Medical Research, University of Belgrade, Serbia, according to the National Law on Animal Welfare, consistent with guidelines for animal research and principles of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Purposes (Official Daily No. 323-07-02569/2018-05).…”

“…Mouse blood was collected from the retro-orbital sinus. Mouse blood cell counts were made using a hemocytometer, as previously described [ 5 ]. Hemoglobin was analyzed with the cyanmethemoglobin method and determined spectrophotometrically (RT-6100 Microplate Reader, Rayto, Shenzhen, China), while hematocrit was calculated after brief centrifugation.…”

“…Plasma nitrite (NO 2 -) and nitrate (NO 3 ) levels were measured by the Griess method, as previously described [ 5 ]. Briefly, 100 µL of each sample and each standard were pipetted into separate wells.…”

“…Mononuclear cells were separated from the bone marrow cells with lymphocyte separation medium (LSM, Capricorn Scientific, Ebsdorfergrund, Germany) according to the manufacturer’s instruction. For cytoplasmatic staining, mice bone marrow cells as well as CFU-E and BFU-E / CFU-GM colonies from methylcellulose cultures were collected onto microscope glass slides, as previously described [ 5 ]. Briefly, after fixation and incubation with the anti-neuronal NOS (nNOS, cat no.…”

“…In preclinical in vitro studies, hydroxyurea increases endothelial cell production of NO by stimulating endothelial NO synthase (eNOS) [ 3 ], while the later study showed that hydroxyurea enhances nitrite production only in combination with heme [ 4 ]. In addition, hydroxyurea regulation of erythroid cell proliferation and apoptosis is NOS dependent [ 5 ]. Hydroxyurea in vitro decreases the growth of erythroid colonies with the participation of NOS [ 6 , 7 ].…”