Low values of 5‐hydroxymethylcytosine (5hmC), the “sixth base,” are associated with anaplasia in human brain tumors

Abstract

5-Methylcytosine (5mC) in genomic DNA has important epigenetic functions in embryonic development and tumor biology. 5-Hydroxymethylcytosine (5hmC) is generated from 5mC by the action of the TET (Ten-Eleven-Translocation) enzymes and may be an intermediate to further oxidation and finally demethylation of 5mC. We have used immunohistochemistry (IHC) and isotope-based liquid chromatography mass spectrometry (LC-MS) to investigate the presence and distribution of 5hmC in human brain and brain tumors. In the normal adult brain, IHC identified 61.5% 5hmC positive cells in the cortex and 32.4% 5hmC in white matter (WM) areas. In tumors, positive staining of cells ranged from 1.1% in glioblastomas (GBMs) (WHO Grade IV) to 8.9% in Grade I gliomas (pilocytic astrocytomas). In the normal adult human brain, LC-MS also showed highest values in cortical areas (1.17% 5hmC/dG [deoxyguanosine]), in the cerebral WM we measured around 0.70% 5hmC/dG. 5hmC levels were related to tumor differentiation, ranging from lowest values of 0.078% 5hmC/dG in GBMs (WHO Grade IV) to 0.24% 5hmC/dG in WHO Grade II diffuse astrocytomas. 5hmC measurements were unrelated to 5mC values. We find that the number of 5hmC positive cells and the amount of 5hmC/dG in the genome that has been proposed to be related to pluripotency and lineage commitment in embryonic stem cells is also associated with brain tumor differentiation and anaplasia.Epigenetics, the study of mechanisms that control gene expression (in a potentially heritable way), may be the most rapidly expanding field in tumor biology. On a molecular level, (i) DNA methylation, i.e., covalent modification of cytosine bases resulting in 5-methylcytosine (5mC), (ii) histone modifications and (iii) nucleosome positioning are regarded as the driving epigenetic mechanisms. They are fundamental to the regulation of many cellular processes, including gene and micro RNA expression, DNA-protein interactions, suppression of transposable element mobility, cellular differentiation, embryogenesis, X-chromosome inactivation and genomic imprinting.In tumor biology, DNA methylation is the best-studied epigenetic change. Epigenetic silencing of O 6 -methylguanine DNA-methyltransferase (MGMT) 1 has been described as a strong predictive factor of treatment response to chemotherapy with alkylating agents of glioblastoma (GBM) and anaplastic astrocytoma (AA). 2 Methylation of CpG islands in the MGMT promoter with ensuing repression of MGMT transcriptional activity is generally viewed as the cause for this correlation. 3 Oxidation of 5mC leading to 5-hydroxymethylcytosine (5hmC) has been identified as a new epigenetic phenomenon in mouse Purkinje cells. 4 Three possible modes of action of 5hmC were discussed. (i) It might influence chromatin structure and local transcriptional activity by recruiting selective 5hmC-binding proteins or excluding 5mC-binding proteins.(ii) Conversion of 5mC to 5hmC might facilitate passive DNA demethylation by excluding the maintenance DNMT1, which recognizes 5hmC poorly. (iii) 5hmC may...

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

“…On the other hand, 5-hmC was reduced by about 3-fold in the tumors of GC cases. In line with recent studies [9,26], our data also show that 5-hmC values varied widely among individuals. P values were analyzed using a two-tailed Pearson correlation.…”

“…and 2-oxoglutarate (2-OG), and 2-OG is dependent on the activity of isocitrate dehydrogenase IDH1 and IDH2 [5,6] ( Figure S1). The loss of 5-hydroxymethylcytosine and the down-regulation of the TET family and IDH2 have been found in human cancers [7][8][9][10][11], but comprehensive evaluations of the modified cytosines and the enzymes that are involved have remained largely unperformed and most previous studies have used dot blot analyses as a means of measuring 5-hydroxymethyldeoxycytidine (5-hmC), which is at best a semi-quantitative method. Because dot blot analysis is based on the antigen-antibody reaction direct assessments of the absolute amount of 5-mC, 5-hmC, 5-fC, and 5-caC are not possible.…”

The potential role of O-GlcNAc modification in cancer epigenetics

“…Jin et al showed tumor-associated loss of 5hmC for lung, brain, breast, liver, kidney, prostate, intestine, and uterus cancers and melanoma [109]. Reduction of 5hmC in different types of solid tumors was confirmed in many other studies [110][111][112][113][114]. Although decreased 5hmC seems to be a common feature of cancers, there is no straight link with TET expression or Brought to you by | MIT Libraries Authenticated Download Date | 5/11/18 7:43 PM mutation levels and aberrant methylation.…”

Chromatin Memory in the Development of Human Cancers

“…The conversion of 5mC to 5-hydroxy-methylcytosine (5hmC) in mammalian cells by methylcytosine dioxygenase Ten-Eleven Translocation (TET) is thought to be an important procedure during active demethylation process [23]. 5hmc has been recognized as the "sixth base" in the genome because of its distinctive epigenetic role [24].…”

“…Brain is one of the organs that with 5hmC presence at high levels. In terms of cancers developed in the central nervous system, it is observed that 5hmC levels decreased by clinical grades of the tumors; meaning high numbers of 5hmC positive cells in WHO grade I gliomas, fewer in grades II and III, and the least number of 5hmC positive cells in grade IV gliomas [24,45].…”