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Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks
BACKGROUND: UROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to ve...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656924/ https://www.ncbi.nlm.nih.gov/pubmed/23691160 http://dx.doi.org/10.1371/journal.pone.0064139 |
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author | Johnen, Georg Rozynek, Peter von der Gathen, Yvonne Bryk, Oleksandr Zdrenka, Ricarda Johannes, Christian Weber, Daniel G. Igwilo-Okuefuna, O′Brien Raiko, Irina Hippler, Jörg Brüning, Thomas Dopp, Elke |
author_facet | Johnen, Georg Rozynek, Peter von der Gathen, Yvonne Bryk, Oleksandr Zdrenka, Ricarda Johannes, Christian Weber, Daniel G. Igwilo-Okuefuna, O′Brien Raiko, Irina Hippler, Jörg Brüning, Thomas Dopp, Elke |
author_sort | Johnen, Georg |
collection | PubMed |
description | BACKGROUND: UROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity. METHODS: UROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied. RESULTS: All tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly. CONCLUSIONS: It is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments. |
format | Online Article Text |
id | pubmed-3656924 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-36569242013-05-20 Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks Johnen, Georg Rozynek, Peter von der Gathen, Yvonne Bryk, Oleksandr Zdrenka, Ricarda Johannes, Christian Weber, Daniel G. Igwilo-Okuefuna, O′Brien Raiko, Irina Hippler, Jörg Brüning, Thomas Dopp, Elke PLoS One Research Article BACKGROUND: UROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity. METHODS: UROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied. RESULTS: All tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly. CONCLUSIONS: It is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments. Public Library of Science 2013-05-17 /pmc/articles/PMC3656924/ /pubmed/23691160 http://dx.doi.org/10.1371/journal.pone.0064139 Text en © 2013 Johnen 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, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Johnen, Georg Rozynek, Peter von der Gathen, Yvonne Bryk, Oleksandr Zdrenka, Ricarda Johannes, Christian Weber, Daniel G. Igwilo-Okuefuna, O′Brien Raiko, Irina Hippler, Jörg Brüning, Thomas Dopp, Elke Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks |
title | Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks |
title_full | Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks |
title_fullStr | Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks |
title_full_unstemmed | Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks |
title_short | Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks |
title_sort | cross-contamination of a urotsa stock with t24 cells – molecular comparison of different cell lines and stocks |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656924/ https://www.ncbi.nlm.nih.gov/pubmed/23691160 http://dx.doi.org/10.1371/journal.pone.0064139 |
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