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. and Martinello-Wilks, Rosetta (2011) Evaluation of cell cycle arrest in estrogen responsive MCF-7 breast cancer cells: Pitfalls of the MTS assay. PLoS ONE, 6 6: e20623.1-e20623.8. doi:10.1371/journal.pone.0020623


Author McGowan, Eileen M.
Alling, Nikki
Jackson, Elise A.
Yagoub, Daniel
Haass, Nikolas K.
Allen, John D.
Martinello-Wilks, Rosetta
Title Evaluation of cell cycle arrest in estrogen responsive MCF-7 breast cancer cells: Pitfalls of the MTS assay
Journal name PLoS ONE   Check publisher's open access policy
ISSN 1932-6203
Publication date 2011-01-01
Year available 2011
Sub-type Article (original research)
DOI 10.1371/journal.pone.0020623
Open Access Status DOI
Volume 6
Issue 6
Start page e20623.1
End page e20623.8
Total pages 8
Place of publication San Francisco, CA United States
Publisher Public Library of Science
Language eng
Subject 1100 Agricultural and Biological Sciences
1300 Biochemistry, Genetics and Molecular Biology
2700 Medicine
Abstract 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.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: UQ Diamantina Institute Publications
 
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Created: Sat, 31 May 2014, 03:48:27 EST by Nikolas Haass on behalf of UQ Diamantina Institute