title: Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.
abstract: OBJECTIVE
  To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T).
  
  
  METHODS
  We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA.
  
  
  METHODS
  The cells were cultured with or without glutamine or GSH.
  
  
  METHODS
  ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting.
  
  
  RESULTS
  While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and cultured without glutamine showed higher levels of ROS and IL-8 than those transfected with negative control siRNA; increased levels of ROS and IL-8 were suppressed by the treatment of glutamine.
  
  
  CONCLUSIONS
  Glutamine deprivation induces ROS production, NF-κB activation, and IL-8 expression as well as a reduction in GSH in A-T fibroblasts, all of which are attenuated by glutamine supplementation.
authors.affiliation: Department of Food and Nutrition, Brain Korea 21 PLUS Project, College of Human Ecology, Yonsei University, Seoul, 120-749, Korea.
authors.forename: Min-Hyun
authors.initials: MH
authors.lastname: Kim
authors.forename: Aryung
authors.initials: A
authors.lastname: Kim
authors.forename: Ji Hoon
authors.initials: JH
authors.lastname: Yu
authors.forename: Joo Weon
authors.initials: JW
authors.lastname: Lim
authors.forename: Hyeyoung
authors.initials: H
authors.lastname: Kim
mesh_terms: D000818:Animals; D001260:Ataxia Telangiectasia / Q000276:immunology / Q000378:metabolism*; D064007:Ataxia Telangiectasia Mutated Proteins / Q000502:physiology; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism*; D005973:Glutamine / Q000502:physiology*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D016209:Interleukin-8; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D005973:Glutamine; D004247:DNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D005978:Glutathione