Quantification of the sixth DNA base 5-hydroxymethylcytosine in colorectal cancer tissue and C-26 cell line.

BACKGROUND DNA methylation at the five position of cytosine is well recognized as an important epigenetic modification in human health and disease. Recent evidences demonstrated that 5-methylcytosine (5-mC) by the TET family of enzymes can be converted to 5-hydroxymethylcytosine (5-hmC). Here, we use an ultrasensitive and accurate isotope-based LC-MS/MS method to precisely determine the levels of 5-hmC and 5-mC in colorectal cancer and the C-26 colon adenocarcinoma cell line. RESULTS Our data showed that 5-hmC content is significantly reduced (approximately sixfold) in colorectal cancer as compared with adjacent normal tissue. Similarly, the ratio of 5-hmC to 5-mC dropped from 0.054 ± 0.005 in normal tissues, to 0.011 ± 0.002 in cancer. CONCLUSION The analysis of 5-hmC levels and the ratio of 5-hmC:5-mC during tumor progression might provide insight into the role of this modification in cellular immortalization and transformation.

[1]  U. Schüller,et al.  Low values of 5‐hydroxymethylcytosine (5hmC), the “sixth base,” are associated with anaplasia in human brain tumors , 2012, International journal of cancer.

[2]  Xiaodong Cheng,et al.  Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation , 2012, Nucleic acids research.

[3]  Keyuan Zhou,et al.  Simultaneous Determination of Global DNA Methylation and Hydroxymethylation Levels by Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry , 2012, Journal of biomolecular screening.

[4]  G. Schackert,et al.  5-Hydroxymethylcytosine is strongly depleted in human cancers but its levels do not correlate with IDH1 mutations. , 2011, Cancer research.

[5]  C. Iacobuzio-Donahue,et al.  Global 5-hydroxymethylcytosine content is significantly reduced in tissue stem/progenitor cell compartments and in human cancers , 2011, Oncotarget.

[6]  Keyuan Zhou,et al.  Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry. , 2011, Analytical biochemistry.

[7]  R. Klose,et al.  The oncometabolite 2‐hydroxyglutarate inhibits histone lysine demethylases , 2011, EMBO reports.

[8]  Riitta Lahesmaa,et al.  Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. , 2011, Cell stem cell.

[9]  Bin Wang,et al.  Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. , 2011, Cancer cell.

[10]  P. Jin,et al.  Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine , 2011, Nature Biotechnology.

[11]  M. Biel,et al.  Tissue Distribution of 5-Hydroxymethylcytosine and Search for Active Demethylation Intermediates , 2010, PloS one.

[12]  L. Aravind,et al.  Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 , 2010, Nature.

[13]  H. Leonhardt,et al.  Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA , 2010, Nucleic acids research.

[14]  A. Baccarelli,et al.  Development and validation of a gas chromatography/mass spectrometry method for the assessment of genomic DNA methylation. , 2009, Rapid communications in mass spectrometry : RCM.

[15]  Z. Liutkevičiūtė,et al.  Cytosine-5-methyltransferases add aldehydes to DNA. , 2009, Nature chemical biology.

[16]  David R. Liu,et al.  Conversion of 5-Methylcytosine to 5- Hydroxymethylcytosine in Mammalian DNA by the MLL Partner TET1 , 2009 .

[17]  N. Heintz,et al.  The Nuclear DNA Base 5-Hydroxymethylcytosine Is Present in Purkinje Neurons and the Brain , 2009, Science.

[18]  M. Esteller.  Epigenetics in cancer. , 2008, The New England journal of medicine.

[19]  Peter A. Jones,et al.  Cancer epigenetics: modifications, screening, and therapy. , 2008, Annual review of medicine.

[20]  Peter A. Jones,et al.  The Epigenomics of Cancer , 2007, Cell.

[21]  Fabio Garofolo,et al.  Bioanalytical Method Validation , 2004 .

[22]  C. Maniatopoulos,et al.  Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats , 1988, Cell and Tissue Research.

[23]  A. Bird,et al.  Oxidative damage to methyl-CpG sequences inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2). , 2004, Nucleic acids research.

[24]  A. Niveleau,et al.  Evaluation of global DNA hypomethylation in human colon cancer tissues by immunohistochemistry and image analysis , 2000, Gut.

[25]  K. Bojanowski,et al.  The presence of 5-hydroxymethylcytosine in animal deoxyribonucleic acid. , 1972, The Biochemical journal.