title: Decitabine induces delayed reactive oxygen species (ROS) accumulation in leukemia cells and induces the expression of ROS generating enzymes.
abstract: OBJECTIVE
  Azanucleoside DNA methyltransferase (DNMT) inhibitors are currently approved by the U.S. Food and Drug Administration for treatment of myelodysplastic syndrome. The relative contributions of DNMT inhibition and other off-target effects to their clinical efficacy remain unclear. Data correlating DNA methylation reversal and clinical response have been conflicting. Consequently, it is necessary to investigate so-called off-target effects and their impact on cell survival and differentiation.
  
  
  METHODS
  Flow cytometry was used for cell cycle, apoptosis, and reactive oxygen species (ROS) accumulation analysis. Gene expression analysis was performed using real-time PCR. DNA methylation was detected by methylation-specific PCR. Mitochondrial membrane potential was analyzed using JC-1 dye staining. Western blotting was used for quantitative protein expression analysis.
  
  
  RESULTS
  5-Aza-2'-deoxycytidine (DAC) induced cell-cycle arrest and apoptosis in leukemia cells. p53 expression was dispensable for DAC-induced apoptosis. DAC induced delayed ROS accumulation in leukemia cells but not in solid tumor cells and p53 expression was dispensable for ROS increase. ROS increase was deoxycytidine kinase dependent, indicating that incorporation of DAC into nuclear DNA is required for ROS generation. ROS accumulation by DAC was caspase-independent and mediated the dissipation of the mitochondrial membrane potential. Concordantly, ROS scavengers diminished DAC-induced apoptosis. DAC induced the expression of different NADPH oxidase isoforms and upregulated Nox4 protein expression in an ATM-dependent manner, indicating the involvement of DNA damage signaling in Nox4 upregulation.
  
  
  CONCLUSIONS
  These data highlight the importance of mechanisms other than DNA cytosine demethylation in modulating gene expression and suggest investigating the relevance of ROS accumulation to the clinical activity of DAC.
authors.affiliation: Authors' Affiliations: Department of Pharmaceutical Sciences, Albany College of Pharmacy, Colchester, Vermont; and The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Tamer E
authors.initials: TE
authors.lastname: Fandy
authors.forename: Anchalee
authors.initials: A
authors.lastname: Jiemjit
authors.forename: Manjusha
authors.initials: M
authors.lastname: Thakar
authors.forename: Paulette
authors.initials: P
authors.lastname: Rhoden
authors.forename: Lauren
authors.initials: L
authors.lastname: Suarez
authors.forename: Steven D
authors.initials: SD
authors.lastname: Gore
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism; D059447:Cell Cycle Checkpoints; D045744:Cell Line, Tumor; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D019175:DNA Methylation; D000077209:Decitabine; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects*; D055785:Gene Knockdown Techniques; D006801:Humans; D007938:Leukemia / Q000201:enzymology* / Q000235:genetics*; D020782:Matrix Metalloproteinases / Q000378:metabolism; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D000975:Antioxidants; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D000077209:Decitabine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D020169:Caspases; D020782:Matrix Metalloproteinases; D001374:Azacitidine