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The cyclooxygenase-2 (COX-2) enzyme and its pro-inflammatory products, prostaglandins and eicosanoids have been implicated in several inflammatory diseases such as atherosclerosis, renal dysfunction and the inhibition of insulin secretion related to Type 1 diabetes. These products are believed to mediate the adhesion to the endothelium and transmigration of activated monocytes into the subendothelial space at the onset of diabetic atherosclerosis. The sequence of the human COX-2 gene is known and several cis-acting regulatory elements have been identified. In this study we have undertaken a DNA-Affinity Capture-μLC/MS/MS analysis of the DNA-protein and protein-protein interactions at the human COX-2 gene promoter to determine the proteins responsible for the upregulation of the gene under diabetic conditions. Two double stranded, 5’ biotinylated DNA capture molecules were employed for the analysis (-580 to -331 and -280 to +1). Four picomoles of each of DNA capture molecule are incubated separately with two THP-1 peripheral blood monocyte protein extracts. The test extract is derived from monocytes grown in the presence of 20 mmol/l D-glucose for 24 hours prior to extraction. The control extract was grown without high glucose. The bound proteins are resolved by 1D SDS-PAGE. The entire sample lane is cut into 4 mm bands, reduced and alkylated and digested with Trypsin overnight. Extracted tryptic peptides were analyzed using an Agilent 1100 Series LC/MSD Trap SL Mass Spectrometer with a nanoelectrospray ion source. Initial 1D SDS-PAGE experiments indicated differential protein binding between the high glucose and control extracts on both DNA capture molecules. Tandem electrospray mass spectrometry has confirmed this observation and identified the proteins responsible differential DNA binding and complex formation. Also, the specific NF-κB site involved in the transcriptional up-regulation of this promoter has been identified.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]