2-acetylaminofluorene up-regulates rat mdr1b expression through generating reactive oxygen species that activate NF-kappa B pathway

J Biol Chem. 2001 Jan 5;276(1):413-20. doi: 10.1074/jbc.M004551200.

Abstract

Overexpression of multidrug resistance genes and their encoded P-glycoproteins is a major mechanism for the development of multidrug resistance in cancer cells. The hepatocarcinogen 2-acetylaminofluorene (2-AAF) efficiently activates rat mdr1b expression. However, the underlying mechanisms are largely unknown. In this study, we demonstrated that a NF-kappa B site on the mdr1b promoter was required for this induction. Overexpression of antisense p65 and I kappa B alpha partially abolished the induction. We then delineated the pathway through which 2-AAF activates NF-kappa B. 2-AAF treatment led to the increase of intracellular reactive oxygen species (ROS) which causes activation of IKK kinases, degradation of I kappa B beta (but not I kappa B alpha), and increase in NF-kappa B DNA binding activity. Consistent with the idea that ROS may participate in mdr1b regulation, antioxidant N-acetylcysteine inhibited the induction of mdr1b by 2-AAF. Overproduction of a physiological antioxidant glutathione (GSH) blocked the activation of IKK kinase complex and NF-kappa B DNA binding. Based on these results, we conclude that 2-AAF up-regulates mdr1b through the generation of ROS, activation of IKK kinase, degradation of I kappa B beta, and subsequent activation of NF-kappa B. This is the first report that reveals the specific cis-elements and signaling pathway responsible for the induction of mdr1b by the chemical carcinogen 2-AAF.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 2-Acetylaminofluorene / pharmacology*
  • ATP Binding Cassette Transporter, Subfamily B / genetics*
  • ATP Binding Cassette Transporter, Subfamily B / metabolism
  • ATP-Binding Cassette Sub-Family B Member 4
  • Acetylcysteine / pharmacology
  • Animals
  • Carcinogens / pharmacology*
  • DNA / genetics
  • DNA / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Enzyme Activation / drug effects
  • Flow Cytometry
  • Gene Expression Regulation / drug effects*
  • Genes, Reporter
  • Hydrogen Peroxide / pharmacology
  • I-kappa B Kinase
  • I-kappa B Proteins*
  • NF-kappa B / metabolism*
  • Oxidative Stress / drug effects
  • Promoter Regions, Genetic / genetics
  • Protein Serine-Threonine Kinases / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Rats
  • Reactive Oxygen Species / metabolism*
  • Signal Transduction / drug effects
  • Transfection
  • Tumor Cells, Cultured
  • Up-Regulation / drug effects

Substances

  • ATP Binding Cassette Transporter, Subfamily B
  • Carcinogens
  • DNA-Binding Proteins
  • I kappa B beta protein
  • I-kappa B Proteins
  • NF-kappa B
  • RNA, Messenger
  • Reactive Oxygen Species
  • DNA
  • 2-Acetylaminofluorene
  • Hydrogen Peroxide
  • Protein Serine-Threonine Kinases
  • I-kappa B Kinase
  • Acetylcysteine