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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
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 intermedia...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708640/ https://www.ncbi.nlm.nih.gov/pubmed/29190698 http://dx.doi.org/10.1371/journal.pone.0188856 |
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author | Foksinski, Marek Zarakowska, Ewelina Gackowski, Daniel Skonieczna, Magdalena Gajda, Karolina Hudy, Dorota Szpila, Anna Bialkowski, Karol Starczak, Marta Labejszo, Anna Czyz, Jaroslaw Rzeszowska-Wolny, Joanna Olinski, Ryszard |
author_facet | Foksinski, Marek Zarakowska, Ewelina Gackowski, Daniel Skonieczna, Magdalena Gajda, Karolina Hudy, Dorota Szpila, Anna Bialkowski, Karol Starczak, Marta Labejszo, Anna Czyz, Jaroslaw Rzeszowska-Wolny, Joanna Olinski, Ryszard |
author_sort | Foksinski, Marek |
collection | PubMed |
description | 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 TET1, TET2, TET3, SMUG1, and TDG 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. |
format | Online Article Text |
id | pubmed-5708640 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-57086402017-12-15 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 Foksinski, Marek Zarakowska, Ewelina Gackowski, Daniel Skonieczna, Magdalena Gajda, Karolina Hudy, Dorota Szpila, Anna Bialkowski, Karol Starczak, Marta Labejszo, Anna Czyz, Jaroslaw Rzeszowska-Wolny, Joanna Olinski, Ryszard PLoS One Research Article 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 TET1, TET2, TET3, SMUG1, and TDG 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. Public Library of Science 2017-11-30 /pmc/articles/PMC5708640/ /pubmed/29190698 http://dx.doi.org/10.1371/journal.pone.0188856 Text en © 2017 Foksinski et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Foksinski, Marek Zarakowska, Ewelina Gackowski, Daniel Skonieczna, Magdalena Gajda, Karolina Hudy, Dorota Szpila, Anna Bialkowski, Karol Starczak, Marta Labejszo, Anna Czyz, Jaroslaw Rzeszowska-Wolny, Joanna Olinski, Ryszard 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 |
title | 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 |
title_full | 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 |
title_fullStr | 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 |
title_full_unstemmed | 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 |
title_short | 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 |
title_sort | 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 |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708640/ https://www.ncbi.nlm.nih.gov/pubmed/29190698 http://dx.doi.org/10.1371/journal.pone.0188856 |
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