14] Assessing oxygen radicals as mediators in activation of inducible eukaryotic transcription factor NF-κB

Abstract

Publisher Summary The eukaryotic transcription factor system specifically activated by peroxides is NF- κ B. Micromolar concentrations of H 2 O 2 can mobilize the sequestered cytoplasmic form of NF- κ B in cultured cells. This involves release of the regulatory subunit I κ B from a heterodimer of DNA-binding p50 and p65 (also called Rel-A) subunits and nuclear translocation of p50-p65. Oxidants activate protein kinases that trigger dissociation of the NF-KB-I κ B. Additional evidence that NF- κ B is an oxidative stress-responsive transcription factor comes from the inhibitory effects of various structurally unrelated antioxidants on the activation of NF- κ B in response to many diverse stimuli. This chapter describes how the activation of NF- κ B by oxidants and the inhibitory effects of antioxidants on activation of NF- κ B are investigated using intact cultured cells. The procedures described in the chapter are particularly useful in achieving two goals. The first goal is to find novel inhibitors of NF- κ B that may be potent antioxidative drugs interfering with the release of I κ B from the cytoplasmic complex of NF- κ B. The second goal is to understand molecular mechanisms underlying the oxidative stress response in higher eukaryotes—that is, to determine what molecules sense a disturbance of the intracellular levels of reactive oxygen intermediates (ROIs) and how they transmit their signals to the nucleus where genes are newly transcribed.