title: Chromatin immunoprecipitation analysis of bortezomib-mediated inhibition of NFκB recruitment to IL-1β and TNFα gene promoters in human macrophages.
abstract: Interleukin-1β (IL-1) and tumor necrosis factor-α (TNF) are important pro-inflammatory cytokines involved in the mediation of the immune response, inflammation, tissue repair, and tumor progression. Regulation of IL-1 and TNF expression is mediated at the level of transcription by the transcription factor NFκB. Inhibition of NFκB activity by the proteasome inhibitor bortezomib (BZ) has been used as a frontline therapy in multiple myeloma and other hematological malignancies. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the NFκB recruitment to endogenous IL-1 and TNF promoters in BZ-treated human macrophages. Corresponding to the BZ-suppressed mRNA levels of IL-1 and TNF, we show that BZ inhibits p65 NFκB recruitment to IL-1 and TNF promoters. This study specifically uses U937 macrophages, but the protocol could be easily modified to analyze the regulation of NFκB recruitment in other cell types.
authors.affiliation: Department of Biology, St. John's University, 8000 Utopia Parkway, Queens, NY, 11439, USA.
authors.forename: Shannon
authors.initials: S
authors.lastname: Sanacora
authors.forename: Tzu-Pei
authors.initials: TP
authors.lastname: Chang
authors.forename: Ivana
authors.initials: I
authors.lastname: Vancurova
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D006801:Humans; D053583:Interleukin-1beta / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011719:Pyrazines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D001897:Boronic Acids; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011719:Pyrazines; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C082161:lipopolysaccharide A; D000069286:Bortezomib; D013755:Tetradecanoylphorbol Acetate