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Levels of oxidative DNA damage are low in ex vivo engineered human limbal epithelial tissue

PURPOSE: To examine levels of oxidative DNA base damage and expression of selected genes and proteins related to DNA damage repair in human limbal epithelium engineered ex vivo. METHODS: Cells were expanded from limbal tissue on cell culture‐treated inserts in medium containing fetal bovine serum, r...

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Autores principales: Lorenzo, Yolanda, Haug Berg, Kristiane, Ringvold, Amund, Petrovski, Goran, Moe, Morten C., Collins, Andrew, Nicolaissen, Bjørn
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667899/
https://www.ncbi.nlm.nih.gov/pubmed/30239138
http://dx.doi.org/10.1111/aos.13811
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author Lorenzo, Yolanda
Haug Berg, Kristiane
Ringvold, Amund
Petrovski, Goran
Moe, Morten C.
Collins, Andrew
Nicolaissen, Bjørn
author_facet Lorenzo, Yolanda
Haug Berg, Kristiane
Ringvold, Amund
Petrovski, Goran
Moe, Morten C.
Collins, Andrew
Nicolaissen, Bjørn
author_sort Lorenzo, Yolanda
collection PubMed
description PURPOSE: To examine levels of oxidative DNA base damage and expression of selected genes and proteins related to DNA damage repair in human limbal epithelium engineered ex vivo. METHODS: Cells were expanded from limbal tissue on cell culture‐treated inserts in medium containing fetal bovine serum, recombinant growth factors, hormones and cholera toxin (COM) and in medium with human serum as the single growth‐promoting additive (HS). Cells were analysed after two, three and four weeks in culture for DNA strand breaks and oxidized purine bases (Comet assay using the enzyme formamidopyrimidine DNA glycosylase, Fpg) and for expression of DNA repair enzymes APE1, OGG1 and Polβ by in situ hybridization (ISH) and by immunohistochemistry (IHC). RESULTS: Levels of strand breaks were substantial while levels of net Fpg‐sensitive sites (8‐oxoguanine and ring‐opened FaPy bases) were relatively low in cells engineered in COM and in HS. Both types of medium were found to support expression of base excision repair (BER) enzymes APE1, OGG1 and Polβ at the gene level. At the protein level, expression of APE1 and OGG1 was noticeable in both conditions while expression of Polβ was low. CONCLUSION: Our findings indicate low levels of oxidative stress and/or efficient DNA purine base damage repair in human limbal epithelium engineered in a medium with human serum as the single growth‐promoting additive as well as in traditional medium with xenobiotics.
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spelling pubmed-66678992019-08-06 Levels of oxidative DNA damage are low in ex vivo engineered human limbal epithelial tissue Lorenzo, Yolanda Haug Berg, Kristiane Ringvold, Amund Petrovski, Goran Moe, Morten C. Collins, Andrew Nicolaissen, Bjørn Acta Ophthalmol Original Articles PURPOSE: To examine levels of oxidative DNA base damage and expression of selected genes and proteins related to DNA damage repair in human limbal epithelium engineered ex vivo. METHODS: Cells were expanded from limbal tissue on cell culture‐treated inserts in medium containing fetal bovine serum, recombinant growth factors, hormones and cholera toxin (COM) and in medium with human serum as the single growth‐promoting additive (HS). Cells were analysed after two, three and four weeks in culture for DNA strand breaks and oxidized purine bases (Comet assay using the enzyme formamidopyrimidine DNA glycosylase, Fpg) and for expression of DNA repair enzymes APE1, OGG1 and Polβ by in situ hybridization (ISH) and by immunohistochemistry (IHC). RESULTS: Levels of strand breaks were substantial while levels of net Fpg‐sensitive sites (8‐oxoguanine and ring‐opened FaPy bases) were relatively low in cells engineered in COM and in HS. Both types of medium were found to support expression of base excision repair (BER) enzymes APE1, OGG1 and Polβ at the gene level. At the protein level, expression of APE1 and OGG1 was noticeable in both conditions while expression of Polβ was low. CONCLUSION: Our findings indicate low levels of oxidative stress and/or efficient DNA purine base damage repair in human limbal epithelium engineered in a medium with human serum as the single growth‐promoting additive as well as in traditional medium with xenobiotics. John Wiley and Sons Inc. 2018-09-21 2018-12 /pmc/articles/PMC6667899/ /pubmed/30239138 http://dx.doi.org/10.1111/aos.13811 Text en © 2018 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
spellingShingle Original Articles
Lorenzo, Yolanda
Haug Berg, Kristiane
Ringvold, Amund
Petrovski, Goran
Moe, Morten C.
Collins, Andrew
Nicolaissen, Bjørn
Levels of oxidative DNA damage are low in ex vivo engineered human limbal epithelial tissue
title Levels of oxidative DNA damage are low in ex vivo engineered human limbal epithelial tissue
title_full Levels of oxidative DNA damage are low in ex vivo engineered human limbal epithelial tissue
title_fullStr Levels of oxidative DNA damage are low in ex vivo engineered human limbal epithelial tissue
title_full_unstemmed Levels of oxidative DNA damage are low in ex vivo engineered human limbal epithelial tissue
title_short Levels of oxidative DNA damage are low in ex vivo engineered human limbal epithelial tissue
title_sort levels of oxidative dna damage are low in ex vivo engineered human limbal epithelial tissue
topic Original Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667899/
https://www.ncbi.nlm.nih.gov/pubmed/30239138
http://dx.doi.org/10.1111/aos.13811
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