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Evaluation of cell cycle arrest in estrogen responsive MCF-7 breast cancer cells: pitfalls of the MTS assay.

McGowan EM, Alling N, Jackson EA, Yagoub D, Haass NK, Allen JD, Martinello-Wilks R - PLoS ONE (2011)

Bottom Line: Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation.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.

View Article: PubMed Central - PubMed

Affiliation: Translational Cancer Research Group, School of Medical and Molecular Biosciences, Faculty of Science, University of Technology Sydney, Sydney, New South Wales, Australia. Eileen.Mcgowan@uts.edu.au

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.

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Related in: MedlinePlus

ICI 182780 and p14ARF increase mitochondria activity.Cells were treated with 10 nM ICI 182780 (A) or 5 mM IPTG (B), 48 h post seeding. At 72 h post treatment cells were counted (see inset) and equal number of cells plated in 96 well plates. Mitochondrial activity was measured using the MTS assay. Treatment results were presented as percentage of control (± SE) in column graphs. Each experiment was performed in duplicate at least 3 times.
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pone-0020623-g004: ICI 182780 and p14ARF increase mitochondria activity.Cells were treated with 10 nM ICI 182780 (A) or 5 mM IPTG (B), 48 h post seeding. At 72 h post treatment cells were counted (see inset) and equal number of cells plated in 96 well plates. Mitochondrial activity was measured using the MTS assay. Treatment results were presented as percentage of control (± SE) in column graphs. Each experiment was performed in duplicate at least 3 times.

Mentions: We have shown significant differences between the MTS and SYBR assay readouts when investigating the anti-proliferative effect of ICI 182780 and p14ARF-induction. We therefore determined mitochondrial activity on a cell-cell basis using the MTS assay. On day 3 post-treatment with ICI 182780, IPTG or control, cells were harvested and equal numbers of treated and untreated cells were seeded into 96 well plates and mitochondrial activity determined by MTS assay (Fig. 4). On a comparative cell-cell basis IPTG-treated cells showed a significant (2.6 fold) increase in mitochondrial activity compared to untreated cells (P<0.0001; Fig. 4B). ICI 182780 also resulted in a significantly increased mitochondrial activity (1.6 fold) per cell (P<0.0001; Fig. 4A).


Evaluation of cell cycle arrest in estrogen responsive MCF-7 breast cancer cells: pitfalls of the MTS assay.

McGowan EM, Alling N, Jackson EA, Yagoub D, Haass NK, Allen JD, Martinello-Wilks R - PLoS ONE (2011)

ICI 182780 and p14ARF increase mitochondria activity.Cells were treated with 10 nM ICI 182780 (A) or 5 mM IPTG (B), 48 h post seeding. At 72 h post treatment cells were counted (see inset) and equal number of cells plated in 96 well plates. Mitochondrial activity was measured using the MTS assay. Treatment results were presented as percentage of control (± SE) in column graphs. Each experiment was performed in duplicate at least 3 times.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3108819&req=5

pone-0020623-g004: ICI 182780 and p14ARF increase mitochondria activity.Cells were treated with 10 nM ICI 182780 (A) or 5 mM IPTG (B), 48 h post seeding. At 72 h post treatment cells were counted (see inset) and equal number of cells plated in 96 well plates. Mitochondrial activity was measured using the MTS assay. Treatment results were presented as percentage of control (± SE) in column graphs. Each experiment was performed in duplicate at least 3 times.
Mentions: We have shown significant differences between the MTS and SYBR assay readouts when investigating the anti-proliferative effect of ICI 182780 and p14ARF-induction. We therefore determined mitochondrial activity on a cell-cell basis using the MTS assay. On day 3 post-treatment with ICI 182780, IPTG or control, cells were harvested and equal numbers of treated and untreated cells were seeded into 96 well plates and mitochondrial activity determined by MTS assay (Fig. 4). On a comparative cell-cell basis IPTG-treated cells showed a significant (2.6 fold) increase in mitochondrial activity compared to untreated cells (P<0.0001; Fig. 4B). ICI 182780 also resulted in a significantly increased mitochondrial activity (1.6 fold) per cell (P<0.0001; Fig. 4A).

Bottom Line: Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation.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.

View Article: PubMed Central - PubMed

Affiliation: Translational Cancer Research Group, School of Medical and Molecular Biosciences, Faculty of Science, University of Technology Sydney, Sydney, New South Wales, Australia. Eileen.Mcgowan@uts.edu.au

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.

Show MeSH
Related in: MedlinePlus