Notes: The enzyme, indoleamine 2,3-dioxygenase (IDO), is associated with cancers and appears to protect tumors from T-lymphocyte response. In this study, the authors sought to downregulate IDO expression by targeting a key regulator of IDO expression, STAT1. They assessed the ability of fludarabine, shown previously to prevent STAT1 phosphorylation, to suppress IDO expression and, consequently, activity. However, although the activity of IDO decreased, the level of transcription of IDO was only marginally effected by fludarabine treatment, suggesting inhibition of activity was occurring later. They found that inhibition of the proteasome partially protected IDO from the effects of fludarabine activity. To investigate the role of the proteasome, they used the Proteasome-Glo™ Chymotrypsin-Like Assay with MDA-231 breast cancer cells. The researchers concluded that a proteasome-dependent process regulates IDO stability and hence its activity. (4501)

Notes: Proteasome activity was determined in cells expressing the β2 proteasome subunit using the Proteasome-Glo™ Trypsin-Like Cell-Based Assay. The authors determined the proteasome activity in the cells after exposure to the proteasome inhibitor MG132. Luminescence indicative of proteasome activity was measured using a GloMax^®-Multi Detection System Luminometer. (4195)

Notes: The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase. (3926)