title: Radiation-sensitive tyrosine phosphorylation of cellular proteins: sensitive to changes in GSH content induced by pretreatment with N-acetyl-L-cysteine or L-buthionine-S,R-sulfoximine.
abstract: OBJECTIVE
  At relatively high concentrations, ie., > 20 mM, N-acetyl-L-cysteine (NAC) scavenges reactive oxygen species produced by ionizing radiation in aqueous solution. Therefore, the ability of NAC to block signal transduction reactions in vivo, has lead to the suggestion that ROS are necessary for the normal propagation of these signals. In this paper we investigate the mechanism by which NAC alters signal transduction in whole cells.
  
  
  RESULTS
  Exposing CHO-K1 cells to ionizing radiation results in elevated pp59fyn kinase activity. Moreover, we observe changes in the phosphotyrosine content of multiple cellular proteins, including one prominent phosphotyrosyl protein with a Mr of 85 kDa. Both the radiation-induced changes in pp59fyn kinase activity and the changes in phosphotyrosine content of pp85 were not affected by exposing K1 cells to NAC during the time of irradiation, suggesting that ROS generated extracellularly are not involved in the radiation-induced changes observed in phosphotyrosyl proteins. We also demonstrate that the cell membrane is an effective barrier against negatively charged NAC. Therefore, it seems unlikely that NAC's ability to block signal transduction reactions is related to scavenging of ROS intracellularly. Chronic exposure, ie., 1 h, to 20 mM NAC lead to a twofold elevation in GSH levels and resulted in a 17% decrease in the phosphotyrosine content of pp85 after exposure to 10 Gy. Moreover, pretreatment with L-buthionine-S,R-sulfoximine (BSO) decreased GSH levels and resulted in elevated phosphotyrosine levels in pp85 isolated from irradiated CHO-K1 cells.
  
  
  CONCLUSIONS
  Since many signaling molecules contain redox sensitive cysteine residues that regulate enzyme activity, we suggest that the effects of NAC on radiation-induced signal transduction are due to its ability to alter the intracellular reducing environment, and not related to direct scavenging of ROS.
authors.affiliation: Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
authors.forename: S
authors.initials: S
authors.lastname: Tuttle
authors.forename: A M
authors.initials: AM
authors.lastname: Horan
authors.forename: C J
authors.initials: CJ
authors.lastname: Koch
authors.forename: K
authors.initials: K
authors.lastname: Held
authors.forename: Y
authors.initials: Y
authors.lastname: Manevich
authors.forename: J
authors.initials: J
authors.lastname: Biaglow
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D016466:CHO Cells / Q000378:metabolism / Q000528:radiation effects; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism; D016166:Free Radical Scavengers / Q000378:metabolism / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism* / Q000528:radiation effects; D051076:Proto-Oncogene Proteins c-fyn; D011836:Radiation Tolerance; D011837:Radiation-Protective Agents / Q000494:pharmacology; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D016166:Free Radical Scavengers; D011518:Proto-Oncogene Proteins; D011837:Radiation-Protective Agents; D011838:Radiation-Sensitizing Agents; D017382:Reactive Oxygen Species; D019328:Buthionine Sulfoximine; D006861:Hydrogen Peroxide; D051076:Proto-Oncogene Proteins c-fyn; D005978:Glutathione; D003545:Cysteine; D000111:Acetylcysteine