title: Detection of methylated CDO1 in plasma of colorectal cancer; a PCR study.
abstract: BACKGROUND
  Cysteine biology is important for the chemosensitivity of cancer cells. Our research has focused on the epigenetic silencing of cysteine dioxygenase type 1 (CDO1) in colorectal cancer (CRC). In this study, we describe detection of CDO1 methylation in the plasma of CRC patients using methylation specific PCR (Q-MSP) and extensive analysis of the PCR reaction.
  
  
  METHODS
  DNA was extracted from plasma, and analysed for methylation of the CDO1 gene using Q-MSP. The detection rate of CDO1 gene methylation was calculated and compared with that of diluted DNA extracted from "positive control" DLD1 cells. CDO1 gene methylation in the plasma of 40 CRC patients that were clinicopathologically analysed was then determined.
  
  
  RESULTS
  (1) The cloned sequence analysis detected 93.3% methylation of the promoter CpG islands of the CDO1 gene of positive control DLD1 cells and 4.7% methylation of the negative control HepG2 CDO1 gene. (2) DLD1 CDO1 DNA could not be detected in this assay if the extracted DNA was diluted ∼1000 fold. The more DNA that was used for the PCR reaction, the more effectively it was amplified in Q-MSP. (3) By increasing the amount of DNA used, methylated CDO1 could be clearly detected in the plasma of 8 (20%) of the CRC patients. However, the percentage of CRC patients detected by methylated CDO1 in plasma was lower than that detected by CEA (35.9%) or CA19-9 (23.1%) in preoperative serum. Combination of CEA/CA19-9 plus plasma methylated CDO1 could increase the rate of detection of curable CRC patients (39.3%) as compared to CEA/CA19-9 (25%).
  
  
  CONCLUSIONS
  We have described detection of CDO1 methylation in the plasma of CRC patients. Although CDO1 methylation was not detected as frequently as conventional tumor markers, analysis of plasma CDO1 methylation in combination with CEA/CA19-9 levels increases the detection rate of curable CRC patients.
authors.affiliation: Department of Surgery, Kitasato University Hospital, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa, 252-0375, Japan.
authors.forename: Keishi
authors.initials: K
authors.lastname: Yamashita
authors.affiliation: Department of Surgery, Kitasato University Hospital, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa, 252-0375, Japan.
authors.forename: Mina
authors.initials: M
authors.lastname: Waraya
authors.affiliation: Department of Otolaryngology, Head and Neck Cancer Research Institute, Johns Hopkins University, 1550 Orleans Street, Baltimore, Maryland, 21231, United States of America.
authors.forename: Myoung Sook
authors.initials: MS
authors.lastname: Kim
authors.affiliation: Department of Otolaryngology, Head and Neck Cancer Research Institute, Johns Hopkins University, 1550 Orleans Street, Baltimore, Maryland, 21231, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Sidransky
authors.affiliation: Department of Surgery, Kitasato University Hospital, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa, 252-0375, Japan.
authors.forename: Natsuya
authors.initials: N
authors.lastname: Katada
authors.affiliation: Department of Surgery, Kitasato University Hospital, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa, 252-0375, Japan.
authors.forename: Takeo
authors.initials: T
authors.lastname: Sato
authors.affiliation: Department of Surgery, Kitasato University Hospital, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa, 252-0375, Japan.
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Department of Surgery, Kitasato University Hospital, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa, 252-0375, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Watanabe
mesh_terms: D018395:CA-19-9 Antigen / Q000097:blood*; D002272:Carcinoembryonic Antigen / Q000097:blood*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000097:blood* / Q000235:genetics; D018899:CpG Islands; D050558:Cysteine Dioxygenase / Q000097:blood / Q000235:genetics*; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic
chemical_list: D018395:CA-19-9 Antigen; D002272:Carcinoembryonic Antigen; C579734:CDO1 protein, human; D050558:Cysteine Dioxygenase