Hydroxyurea enhances SMN2 gene expression through nitric oxide release

Abstract

Small molecules that increase full-length survivor motor neuron (SMN) gene transcript are promising therapeutic candidates for spinal muscular atrophy (SMA). Hydroxyurea (HU) has recently been shown to increase full-length SMN transcript in cultured lymphocytes from patients with SMA. We investigate the mechanism by which HU enhances full-length SMN2 gene expression in SMA lymphocytes. Nitric oxide (NO) is a major intracellular metabolite of HU. We test whether NO donors can themselves enhance full-length SMN2 expression. Eighteen cell lines (five type I, five type II, six type III SMA, and two non-SMA controls) were treated with or without NO donors for 48 h. SMA cells treated with HU and three NO donors: two long-acting donors, Deta-NONOate and S-nitrosoglutathione, and one short-acting donor, 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene, resulted in significant increase in full-length SMN2 mRNA. These effects were abolished by co-treatment with an NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide. One short-acting NO donor, S-nitroso-N-acetyl-DL-penicillamine, failed to show significant effect on full-length SMN2 expression, possibly due to high degree of cytotoxicity. These results were observed using both densitometry and quantitative PCR methods. We conclude that HU enhances SMN2 expression through the release of NO. NO donors may themselves be considered as new therapeutic candidates for SMA.

Treatment of spinal muscular atrophy cells with drugs that upregulate SMN expression reveals inter- and intra-patient variability

“…It has recently been suggested that HU enhances SMN2 expression in lymphoblasts through the release of nitric oxide but its mechanism of action on SMN2 genes is as yet unknown. 37 In the present study, the three compounds tended to show a different intra-patient response depending on the tissues under investigation. This can be partially explained by the fact that fibroblasts are primary cultures that grow as monolayers, whereas lymphoblasts are EBV-immortalized cells that grow in suspension.…”

Spinal Muscular Atrophies

“…[80][81][82] A small pilot study of hydroxyurea at 3 different doses for 8 weeks in 33 types II and III patients, however, showed no statistically significant benefit. 81 A randomized, double-blind, placebocontrolled trial failed to show any improvement over an 18-month period.…”

Spinal Muscular Atrophy: A Clinical and Research Update

“…The drug hydroxyurea, approved by the United States Food and Drug Administration, was identified in the course of drug screens, using cell lines from patients with spinal muscular atrophy, to increase the amount of full-length SMN transcript and protein in vitro [101][102][103]. However, a small pilot study of hydroxyurea at three different doses for 8 weeks in 33 patients with types II and III demonstrated no statistically significant benefit [102].…”