title: γ-radiation induces cellular sensitivity and aberrant methylation in human tumor cell lines.
abstract: OBJECTIVE
  Ionizing radiation induces cellular damage through both direct and indirect mechanisms, which may include effects from epigenetic changes. The purpose of this study was to determine the effect of ionizing radiation on DNA methylation patterns that may be associated with altered gene expression.
  
  
  METHODS
  Sixteen human tumor cell lines originating from various cancers were initially tested for radiation sensitivity by irradiating them with γ-radiation in vitro and subsequently, radiation sensitive and resistant cell lines were treated with different doses of a demethylating agent, 5-Aza-2'-Deoxycytidine (5-aza-dC) and a chromatin modifier, Trichostatin-A (TSA). Survival of these cell lines was measured using 3-(4, 5-Dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium (MTT) and clonogenic assays. The effect of radiation on global DNA methylation was measured using reverse phase high performance liquid chromatography (RP-HPLC). The transcription response of methylated gene promoters, from cyclin-dependent kinase inhibitor 2A (p16(INK4a)) and ataxia telangiectasia mutated (ATM) genes, to radiation was measured using a luciferase reporter assay.
  
  
  RESULTS
  γ-radiation resistant (SiHa and MDAMB453) and sensitive (SaOS2 and WM115) tumor cell lines were examined for the relationship between radiation sensitivity and DNA methylation. Treatment of cells with 5-aza-dC and TSA prior to irradiation enhanced DNA strand breaks, G2/M phase arrest, apoptosis and cell death. Exposure to γ-radiation led to global demethylation in a time-dependent manner in tumor cells in relation to resistance and sensitivity to radiation with concomitant activation of p16(INK4a) and ATM gene promoters.
  
  
  CONCLUSIONS
  These results provide important information on alterations in DNA methylation as one of the determinants of radiation effects, which may be associated with altered gene expression. Our results may help in delineating the mechanisms of radiation resistance in tumor cells, which can influence diagnosis, prognosis and eventually therapy for human cancers.
authors.affiliation: Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Karnataka, India.
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
authors.forename: Padmalatha S
authors.initials: PS
authors.lastname: Rai
authors.forename: Raghavendra
authors.initials: R
authors.lastname: Upadhya
authors.lastname: Vishwanatha
authors.forename: K Shama
authors.initials: KS
authors.lastname: Prasada
authors.forename: B S Satish
authors.initials: BS
authors.lastname: Rao
authors.forename: Kapettu
authors.initials: K
authors.lastname: Satyamoorthy
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002453:Cell Cycle / Q000528:radiation effects; D018797:Cell Cycle Proteins / Q000235:genetics; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000528:radiation effects; D053960:DNA Breaks; D019175:DNA Methylation / Q000528:radiation effects*; D004268:DNA-Binding Proteins / Q000235:genetics; D000077209:Decitabine; D005720:Gamma Rays; D019942:Genes, p16; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011836:Radiation Tolerance; D025521:Tumor Suppressor Proteins / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D006877:Hydroxamic Acids; D025521:Tumor Suppressor Proteins; C012589:trichostatin A; D000077209:Decitabine; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D001374:Azacitidine