A marked difference exists in the inducibility of inducible NO synthase (iNOS) between humans and rodents. Although important cis and trans factors in the murine and human iNOS promoters have been characterized using episomal-based approaches, a compelling molecular explanation for why human iNOS is resistant to induction has not been reported. In this study we present evidence that the hyporesponsiveness of the human iNOS promoter is based in part on epigenetic silencing, specifically hypermethylation of CpG dinucleotides and histone H3 lysine 9 methylation. Using bisulfite sequencing, we demonstrated that the iNOS promoter was heavily methylated at CpG dinucleotides in a variety of primary human endothelial cells and vascular smooth muscle cells, all of which are notoriously resistant to iNOS induction. In contrast, in human cell types capable of iNOS induction (e.g., A549 pulmonary adenocarcinoma, DLD-1 colon adenocarcinoma, and primary hepatocytes), the iNOS promoter was relatively hypomethylated. Treatment of human cells, such as DLD-1, with a DNA methyltransferase inhibitor (5-azacytidine) induced global and iNOS promoter DNA hypomethylation. Importantly, 5-azacytidine enhanced the cytokine inducibility of iNOS. Using chromatin immunoprecipitation, we found that the human iNOS promoter was basally enriched with di- and trimethylation of H3 lysine 9 in endothelial cells, and this did not change with cytokine addition. This contrasted with the absence of lysine 9 methylation in inducible cell types. Importantly, chromatin immunoprecipitation demonstrated the selective presence of the methyl-CpG-binding transcriptional repressor MeCP2 at the iNOS promoter in endothelial cells. Collectively, our work defines a role for chromatin-based mechanisms in the control of human iNOS gene expression.

IntroductionAt a fundamental level, patients with acute leukemia do not respond to treatment because the malignant blasts are not eradicated by current chemotherapy. In part, this failure is due to defects in apoptosis pathways. 1 Therefore, agents that overcome roadblocks to apoptosis could be therapeutically useful for this disease.Classically, apoptosis is caused by the activation of caspases, a family of intracellular cysteine proteases that cleave substrates at aspartic acid residues. 2,3 Currently, at least 4 pathways for initiation of caspase activation exist: (1) the mitochondrial pathway with cytochrome c; (2) the death receptor pathway with the tumor necrosis factor (TNF) family of death receptors; (3) direct caspase activation by cytolytic T-cell protease Granzyme B; and (4) a pathway connected to the endoplasmic reticulum. These pathways launch a proteolytic cascade, in which upstream (initiator) caspases cleave and activate downstream (effector) caspases.The inhibitor of apoptosis proteins (IAPs) are a family of endogenous caspase inhibitors that share a common baculoviral IAP repeat (BIR) domain. To date, 8 IAP family members exist in humans. Of these, XIAP is probably the best characterized with respect to its structure and biochemical mechanisms. XIAP inhibits caspases 3, 7, and 9, but not caspases 1, 6, 8, or 10. 4 XIAP contains 3 tandem baculovirus IAP repeat (BIR) domains and a really interesting new gene (RING) domain. The second BIR domain of XIAP (BIR2) inhibits caspases 3 and 7, while the third BIR domain (BIR3) inhibits caspase 9. Through their ability to inhibit caspases, IAPs act as antiapoptotic proteins [5][6][7][8][9][10] and are promising therapeutic targets. Inhibition of XIAP by antisense strategies or peptides that bind and inhibit the BIR3 domain of XIAP sensitizes malignant cells to chemotherapy. [11][12][13][14][15][16][17][18] Based on the knowledge that XIAP directly inhibits active caspase 3, we devised an enzymatic derepression assay to screen for molecules that relieve protease inhibition. Using this assay, we screened combinatorial libraries of chemical compounds and identified active agents based on different pharmacophores. The initial report of these XIAP inhibitors described a series of compounds based on the polyphenylurea pharmacophore including the active compound N- -methyl-NЈ-phenylurea (1396-12) and structural analogues. 19 Corresponding to their activity in the enzymatic assay, active polyphenylurea-based inhibitors but not inactive controls, induced rapid apoptosis of several types of tumor cell lines. 19 We determined that active compounds inhibit XIAP by binding its BIR2 domain at a site distinct from the binding pocket of the endogenous XIAP inhibitor second modulator of apoptotic proteases (SMAC). 20 Given the potential therapeutic utility of IAP inhibition, we tested these chemical IAP inhibitors in cultured leukemia cell lines For personal use only. on June 19, 2019. by guest www.bloodjournal.org From and primary acute myelogenous leukemia (AML) patie...

FLIP may be a suppressor of anoikis and therefore a possible target for antimetastatic therapeutic strategies.