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<p>Abstract</p> <p>Background</p> <p>Regulatory functions of nitric oxide (NO^•) that bypass the second messenger cGMP are incompletely understood. Here, cGMP-independent effects of NO^•on gene expression were globally examined in U937 cells, a human monoblastoid line that constitutively lacks soluble guanylate cyclase. Differentiated U937 cells (>80% in G0/G1) were exposed to S-nitrosoglutathione, a NO^•donor, or glutathione alone (control) for 6 h without or with dibutyryl-cAMP (Bt₂cAMP), and then harvested to extract total RNA for microarray analysis. Bt₂cAMP was used to block signaling attributable to NO^•-induced decreases in cAMP.</p> <p>Results</p> <p>NO^•regulated 110 transcripts that annotated disproportionately to the cell cycle and cell proliferation (47/110, 43%) and more frequently than expected contained AU-rich, post-transcriptional regulatory elements (ARE). Bt₂cAMP regulated 106 genes; cell cycle gene enrichment did not reach significance. Like NO^•, Bt₂cAMP was associated with ARE-containing transcripts. A comparison of NO^•and Bt₂cAMP effects showed that NO^•regulation of cell cycle genes was independent of its ability to interfere with cAMP signaling. Cell cycle genes induced by NO^•annotated to G1/S (7/8) and included E2F1 and p21/Waf1/Cip1; 6 of these 7 were E2F target genes involved in G1/S transition. Repressed genes were G2/M associated (24/27); 8 of 27 were known targets of p21. E2F1 mRNA and protein were increased by NO^•, as was E2F1 binding to E2F promoter elements. NO^•activated p38 MAPK, stabilizing p21 mRNA (an ARE-containing transcript) and increasing p21 protein; this increased protein binding to CDE/CHR promoter sites of p21 target genes, repressing key G2/M phase genes, and increasing the proportion of cells in G2/M.</p> <p>Conclusion</p> <p>NO^•coordinates a highly integrated program of cell cycle arrest that regulates a large number of genes, but does not require signaling through cGMP. In humans, antiproliferative effects of NO^•may rely substantially on cGMP-independent mechanisms. Stress kinase signaling and alterations in mRNA stability appear to be major pathways by which NO^•regulates the transcriptome.</p>