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Robust quantitative assessments of cytosine modifications and changes in the expressions of related enzymes in gastric cancer.

AbstractBACKGROUND:
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.
AuthorsChunping Du, Nobuya Kurabe, Yoshitaka Matsushima, Masako Suzuki, Tomoaki Kahyo, Ippei Ohnishi, Fumihiko Tanioka, Shogo Tajima, Masanori Goto, Hidetaka Yamada, Hong Tao, Kazuya Shinmura, Hiroyuki Konno, Haruhiko Sugimura
JournalGastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association (Gastric Cancer) Vol. 18 Issue 3 Pg. 516-25 (Jul 2015) ISSN: 1436-3305 [Electronic] Japan
PMID25098926 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • 5-formyl-2'-deoxycytidine
  • 5-hydroxymethyl-2'-deoxycytidine
  • DNA-Binding Proteins
  • Enzymes
  • Proto-Oncogene Proteins
  • Deoxycytidine
  • 5-hydroxymethylcytosine
  • 5-Methylcytosine
  • Cytosine
  • 5-methyldeoxycytidine
  • Mixed Function Oxygenases
  • TET1 protein, human
  • TET3 protein, human
  • IDH2 protein, human
  • Isocitrate Dehydrogenase
  • IDH1 protein, human
  • Dioxygenases
  • TET2 protein, human
Topics
  • 5-Methylcytosine (analogs & derivatives)
  • Aged
  • Chromatography, Liquid
  • Cytosine (analogs & derivatives, analysis, metabolism)
  • DNA-Binding Proteins (genetics)
  • Deoxycytidine (analogs & derivatives, analysis, metabolism)
  • Dioxygenases (genetics)
  • Enzymes (genetics, metabolism)
  • Female
  • Gene Expression Regulation, Neoplastic
  • HEK293 Cells
  • Humans
  • Isocitrate Dehydrogenase (genetics)
  • Male
  • Middle Aged
  • Mixed Function Oxygenases
  • Polymorphism, Single Nucleotide
  • Proto-Oncogene Proteins (genetics)
  • Stomach Neoplasms (enzymology, genetics, metabolism)
  • Tandem Mass Spectrometry

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