title: Quantification of 5-methylcytosine, 5-hydroxymethylcytosine and 5-carboxylcytosine from the blood of cancer patients by an enzyme-based immunoassay.
abstract: BACKGROUND
  Genome-wide aberrations of the classic epigenetic modification 5-methylcytosine (5mC), considered the hallmark of gene silencing, has been implicated to play a pivotal role in mediating carcinogenic transformation of healthy cells. Recently, three epigenetic marks derived from enzymatic oxidization of 5mC namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), have been discovered in the mammalian genome. Growing evidence suggests that these novel bases possess unique regulatory functions and may play critical roles in carcinogenesis.
  
  
  METHODS
  To provide a quantitative basis for these rare epigenetic marks, we have designed a biotin-avidin mediated enzyme-based immunoassay (EIA) and evaluated its performance in genomic DNA isolated from blood of patients diagnosed with metastatic forms of lung, pancreatic and bladder cancer, as well as healthy controls. The proposed EIA incorporates spatially optimized biotinylated antibody and a high degree of horseradish-peroxidase (HRP) labeled streptavidin, facilitating signal amplification and sensitive detection.
  
  
  RESULTS
  We report that the percentages of 5mC, 5hmC and 5caC present in the genomic DNA of blood in healthy controls as 1.025±0.081, 0.023±0.006 and 0.001±0.0002, respectively. We observed a significant (p<0.05) decrease in the mean global percentage of 5hmC in blood of patients with malignant lung cancer (0.013±0.003%) in comparison to healthy controls.
  
  
  CONCLUSIONS
  The precise biological roles of these epigenetic modifications in cancers are still unknown but in the past two years it has become evident that the global 5hmC content is drastically reduced in a variety of cancers. To the best of our knowledge, this is the first report of decreased 5hmC content in the blood of metastatic lung cancer patients and the clinical utility of this observation needs to be further validated in larger sample datasets.
authors.affiliation: Bindley Bioscience & Birck Nanotechnology Center, Department of Agricultural & Biological Engineering, Purdue University, 225 South University Street, West Lafayette, IN 47907, USA.
authors.forename: Basudev
authors.initials: B
authors.lastname: Chowdhury
authors.affiliation: Bindley Bioscience & Birck Nanotechnology Center, Department of Agricultural & Biological Engineering, Purdue University, 225 South University Street, West Lafayette, IN 47907, USA; Dept. of Biomedical Laboratory Science, College of Health Science, Eulji University, Seongnam 461-713, Republic of Korea.
authors.forename: Il-Hoon
authors.initials: IH
authors.lastname: Cho
authors.affiliation: Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN 46202, USA.
authors.forename: Noah
authors.initials: N
authors.lastname: Hahn
authors.affiliation: Bindley Bioscience & Birck Nanotechnology Center, Department of Agricultural & Biological Engineering, Purdue University, 225 South University Street, West Lafayette, IN 47907, USA. Electronic address: josephi@purdue.edu.
authors.forename: Joseph
authors.initials: J
authors.lastname: Irudayaraj
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis* / Q000097:blood / Q000031:analogs & derivatives; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood; D004247:DNA / Q000097:blood* / Q000737:chemistry / Q000235:genetics; D044127:Epigenesis, Genetic; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D057230:Limit of Detection; D009369:Neoplasms / Q000097:blood* / Q000235:genetics
chemical_list: C560974:5-carboxylcytosine; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA
keywords: 5-Carboxylcytosine (5caC); 5-Formylcytosine (5fC); 5-Hydroxymethylcytosine (5hmC); 5-Methylcytosine (5mC); Cancer; Enzyme-based Immunoassay (EIA); Epigenetic modifications