title: Mitochondrial DNA 4977-bp deletion correlated with reactive oxygen species production and manganese superoxidedismutase expression in gastric tumor cells.
abstract: BACKGROUND
  Mitochondrial DNA 4977-bp deletion (DeltamtDNA(4977)) was reported in many human neoplasia. However, its biological significance remains to be evaluated and the molecular mechanism needs to be investigated. In this study, we analyzed the frequency of DeltamtDNA(4977) in gastric cancer (GC) cell lines and tissues, as well as reactive oxygen species (ROS) contents and manganese superoxide dismutase (MnSOD) expression levels in GC cell lines to explore its biological significance and molecular mechanism.
  
  
  METHODS
  Semi-quantitative PCR and real-time PCR were used to detect the incidence of DeltamtDNA(4977) in 13 GC cell lines and 272 human gastric tissues (108 GC specimens and the respective adjacent normal tissues, and 56 normal gastric mucosa from non-cancer patients). We further identified intracellular ROS production by flow cytometry and MnSOD expression by semi-quantitative reverse transcription-PCR (RT-PCR) and Western blotting. Statistical analyses were carried out using the Logistic regression analysis and Kaplan-Meier method.
  
  
  RESULTS
  Based on our earlier study, we optimized the PCR amplification condition by reducing the cycle number. In this study, we systematically documented the high incidence of DeltamtDNA(4977) in GC cell lines (10/13, 76.9%), GC tissues (86/108, 79.6%), matched normal tissues (73/108, 67.6%), and normal gastric mucosa of non-cancer patients (29/56, 51.8%). A significantly higher incidence of mutated DeltamtDNA(4977) was observed in GC tissues with respect to the adjacent normal tissues (79.6% vs 67.6%, P = 0.045), and they were both higher than that in normal controls (P < 0.05). Most importantly, we linked the DeltamtDNA(4977) mutations with the expression level of MnSOD and ROS contents. The cell lines containing lower expression level of MnSOD was found to have generally higher frequent DeltamtDNA(4977) and more ROS.
  
  
  CONCLUSIONS
  The decreased anti-oxidative ability, which leads to increased ROS contents, is correlated with the mtDNA damage during gastric carcinogenesis.
authors.affiliation: Laboratory of Molecular Oncology, Beijing Cancer Hospital and Institute, Peking University School of Oncology, Beijing 100142, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Wang
authors.forename: You-yong
authors.initials: YY
authors.lastname: Lü
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004272:DNA, Mitochondrial / Q000235:genetics*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008875:Middle Aged; D009154:Mutation; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*
chemical_list: D004272:DNA, Mitochondrial; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase