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 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

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