title: Robust quantitative assessments of cytosine modifications and changes in the expressions of related enzymes in gastric cancer.
abstract: BACKGROUND
  The rediscovery of 5-hydroxymethylcytosine, the ten-eleven translocation (TET) family, thymine-DNA glycosylase (TDG) and isocitrate dehydrogenase (IDH) have opened new avenues in the study of DNA demethylation pathways in gastric cancer (GC). We performed a comprehensive and robust analysis of these genes and modified cytosines in gastric cancer.
  
  
  METHODS
  Liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) was used to assess 5-methyldeoxycytidine (5-mC), 5-hydroxymethyldeoxycytidine (5-hmC), 5-formyldeoxycytidine (5-fC) and 5-carboxyldeoxycytidine (5-caC) quantitatively in tumorous and non-tumorous regions of GCs; [D2]-5-hmC was used as an internal standard. Expression levels of the genes TET1, TET2, TET3, TDG, IDH1 and IDH2 were measured using a real-time reverse transcription polymerase chain reaction (RT-PCR) and were compared to the clinical attributes of each case. Using HEK293T cells the effects of introducing plasmids containing full-length TET1, TET2, and TET3 and 7 variants of the TET2 catalytic domain were evaluated in terms of their effect on cytosine demethylation.
  
  
  RESULTS
  LC-MS/MS showed that 5-hmC was significantly decreased in tumorous portions. 5-mC was also moderately decreased in tumors, while 5-fC and 5-caC were barely detectable. The expressions of TET1, TET2, TET3, TDG and IDH2, but not IDH1, were notably decreased in GCs, compared with the adjacent non-tumor portion. TET1 expression and the 5-hmC levels determined using LC-MS/MS had a significantly positive correlation and TET1 protein had a greater effect on the increase in 5-hmC than TET2 and TET3 in HEK293T cells.
  
  
  CONCLUSIONS
  The loss of 5-hmC and the down-regulation of TET1-3, TDG and IDH2 were found in GCs. The loss of 5-hmC in GCs was mainly correlated with the down-regulation of TET1.
authors.affiliation: Department of Tumor Pathology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi Ward, Hamamatsu, Shizuoka, 431-3192, Japan.
authors.forename: Chunping
authors.initials: C
authors.lastname: Du
authors.forename: Nobuya
authors.initials: N
authors.lastname: Kurabe
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Matsushima
authors.forename: Masako
authors.initials: M
authors.lastname: Suzuki
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Kahyo
authors.forename: Ippei
authors.initials: I
authors.lastname: Ohnishi
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Tanioka
authors.forename: Shogo
authors.initials: S
authors.lastname: Tajima
authors.forename: Masanori
authors.initials: M
authors.lastname: Goto
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Yamada
authors.forename: Hong
authors.initials: H
authors.lastname: Tao
authors.forename: Kazuya
authors.initials: K
authors.lastname: Shinmura
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Konno
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Sugimura
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000368:Aged; D002853:Chromatography, Liquid; D003596:Cytosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D049308:Dioxygenases / Q000235:genetics; D004798:Enzymes / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics; D008297:Male; D008875:Middle Aged; D006899:Mixed Function Oxygenases; D020641:Polymorphism, Single Nucleotide; D011518:Proto-Oncogene Proteins / Q000235:genetics; D013274:Stomach Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: C402104:5-formyl-2'-deoxycytidine; C540838:5-hydroxymethyl-2'-deoxycytidine; D004268:DNA-Binding Proteins; D004798:Enzymes; D011518:Proto-Oncogene Proteins; D003841:Deoxycytidine; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; C016569:5-methyldeoxycytidine; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human; C578792:TET3 protein, human; C538784:IDH2 protein, human; D007521:Isocitrate Dehydrogenase; C543588:IDH1 protein, human; D049308:Dioxygenases; C541081:TET2 protein, human
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