Levels of 5-mdC and intermediate products of active DNA demethylation in DNA from normal fibroblasts and various malignant cell lines.

<p>(A) Level of 5-mdC. (B) Level of 5-hmdC. (C) Level of 5-fdC. (D) Level of 5-cadC. (E) Level of 5-hmdU.</p

Doubling time of cells.

<p>Doubling time of cells.</p

Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2′-deoxycytidine level in cultured cancerous cell lines

<div><p>Active demethylation of 5-methylcytosine moiety in DNA occurs by its sequential oxidation to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, catalysed by enzymes of the Ten-Eleven Translocation family proteins (TETs 1, 2 and 3). Here we analyzed for the first time all the intermediate products of DNA demethylation pathway in the form of deoxynucleosides (5-methyl-2′-deoxycytidine, 5-(hydroxymethyl)-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxy-2′-deoxycytidine as well as 5-(hydroxymethyl)-2′-deoxyuridine) using automated isotope-dilution online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. DNA was isolated from human malignant cell lines of colon adenocarcinoma (HCT 116), melanoma (Me45), myelogenous leukemia bone marrow blasts (K562), EBV-positive Burkitt’s lymphoma lymphoblasts (Raji), EBV-negative Burkitt’s lymphoma lymphoblasts (male-CA46 and female-ST486), as well as normal neonatal dermal fibroblasts (NHDF-Neo). The expression levels of <i>TET1</i>, <i>TET2</i>, <i>TET3</i>, <i>SMUG1</i>, and <i>TDG</i> genes were also assayed by RT-qPCR. Our results show a global erasure of 5-hydroxymethyl-2′-deoxycytidine and 5-carboxy-2′-deoxycytidine in DNA of cultured cells compared with DNA from primary malignant tissue. Moreover, malignant cells in culture have a quite different DNA epigenetic profile than cultured normal cells, and different types of malignant cells display different and characteristic profiles of DNA epigenetic marks. Similar analyses of a broader spectrum of epigenetic modifications, not restricted to 5-methyl-2′-deoxycytidine, could lead to better understanding of the mechanism(s) responsible for emergence of different types of cancer cells.</p></div

Levels of transcripts of the <i>TETs</i>, <i>SMUG1</i> and <i>TDG</i> genes in various cell lines.

<p>(A) <i>TET1</i>. (B) <i>TET2</i>. (C) <i>TET3</i>. (D) <i>SMUG1</i>. (E) <i>TDG</i>.</p

A positive correlation between 5-(hydroxymethyl)-2′-deoxycytidine and mRNA expression level of <i>TET1</i> gene in malignant cell lines.

<p>A positive correlation between 5-(hydroxymethyl)-2′-deoxycytidine and mRNA expression level of <i>TET1</i> gene in malignant cell lines.</p

Levels of intermediate products of active DNA demethylation in DNA from cultured cells compared with DNA from primary malignant tissue.

<p>(A) Level of 5-hmdC. (B) Level of 5-fdC. (C) Level of 5-cadC. (D) Level of 5-hmdU.</p

MOESM1 of In vivo evidence of ascorbate involvement in the generation of epigenetic DNA modifications in leukocytes from patients with colorectal carcinoma, benign adenoma and inflammatory bowel disease

Additional file 1. Tables S1–S3, Figures S1–S10, additional method

Additional file 2: of Characteristic profiles of DNA epigenetic modifications in colon cancer and its predisposing conditions—benign adenomas and inflammatory bowel disease

Table S2. Transition patterns specific detector settings and sources of standards for analyzed compounds (relative response ratio = area under the peak of qualifierion/area under the peak of quantifier ion). (PDF 82 kb

Additional file 1: of Characteristic profiles of DNA epigenetic modifications in colon cancer and its predisposing conditions—benign adenomas and inflammatory bowel disease

Table S1. Characteristics of antibodies used in immunohistochemical staining. (PDF 499 kb

Additional file 3: of Characteristic profiles of DNA epigenetic modifications in colon cancer and its predisposing conditions—benign adenomas and inflammatory bowel disease

Table S3. Primers and short hydrolysis probes used for TETs and AID mRNA expression analysis. (PDF 442 kb