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Evaluation of Cell Cycle Arrest in Estrogen Responsive MCF-7 Breast Cancer Cells: Pitfalls of the MTS Assay
Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report descri...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3108819/ https://www.ncbi.nlm.nih.gov/pubmed/21673993 http://dx.doi.org/10.1371/journal.pone.0020623 |
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author | McGowan, Eileen M. Alling, Nikki Jackson, Elise A. Yagoub, Daniel Haass, Nikolas K. Allen, John D. Martinello-Wilks, Rosetta |
author_facet | McGowan, Eileen M. Alling, Nikki Jackson, Elise A. Yagoub, Daniel Haass, Nikolas K. Allen, John D. Martinello-Wilks, Rosetta |
author_sort | McGowan, Eileen M. |
collection | PubMed |
description | Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2′-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens. |
format | Online Article Text |
id | pubmed-3108819 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-31088192011-06-13 Evaluation of Cell Cycle Arrest in Estrogen Responsive MCF-7 Breast Cancer Cells: Pitfalls of the MTS Assay McGowan, Eileen M. Alling, Nikki Jackson, Elise A. Yagoub, Daniel Haass, Nikolas K. Allen, John D. Martinello-Wilks, Rosetta PLoS One Research Article Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2′-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens. Public Library of Science 2011-06-03 /pmc/articles/PMC3108819/ /pubmed/21673993 http://dx.doi.org/10.1371/journal.pone.0020623 Text en McGowan 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 McGowan, Eileen M. Alling, Nikki Jackson, Elise A. Yagoub, Daniel Haass, Nikolas K. Allen, John D. Martinello-Wilks, Rosetta Evaluation of Cell Cycle Arrest in Estrogen Responsive MCF-7 Breast Cancer Cells: Pitfalls of the MTS Assay |
title | Evaluation of Cell Cycle Arrest in Estrogen Responsive MCF-7 Breast Cancer Cells: Pitfalls of the MTS Assay |
title_full | Evaluation of Cell Cycle Arrest in Estrogen Responsive MCF-7 Breast Cancer Cells: Pitfalls of the MTS Assay |
title_fullStr | Evaluation of Cell Cycle Arrest in Estrogen Responsive MCF-7 Breast Cancer Cells: Pitfalls of the MTS Assay |
title_full_unstemmed | Evaluation of Cell Cycle Arrest in Estrogen Responsive MCF-7 Breast Cancer Cells: Pitfalls of the MTS Assay |
title_short | Evaluation of Cell Cycle Arrest in Estrogen Responsive MCF-7 Breast Cancer Cells: Pitfalls of the MTS Assay |
title_sort | evaluation of cell cycle arrest in estrogen responsive mcf-7 breast cancer cells: pitfalls of the mts assay |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3108819/ https://www.ncbi.nlm.nih.gov/pubmed/21673993 http://dx.doi.org/10.1371/journal.pone.0020623 |
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