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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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Comparative analysis of MCF-7 cell viability, cell number and mitochondrial activity.Cells were treated with 5 mM IPTG, 10 nM ICI 182780, 5 µM FTY720, or serum deprived (serum free) 48 h post-seeding. Using the Trypan Blue exclusion method cells were harvested and viable cells counted using a haemocytometer at days indicated. MTS and SYBR-DNA assays were performed, as detailed in materials and methods, at days indicated. The treatment results are shown as a percentage of the uninduced vehicle control (±SE) correlating with viable cell number (Trypan blue counts), colorimetric measurement (MTS), and fluorescent intensity (SYBR assay). Each experiment was performed in triplicate at least 3 times with similar results.
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pone-0020623-g001: Comparative analysis of MCF-7 cell viability, cell number and mitochondrial activity.Cells were treated with 5 mM IPTG, 10 nM ICI 182780, 5 µM FTY720, or serum deprived (serum free) 48 h post-seeding. Using the Trypan Blue exclusion method cells were harvested and viable cells counted using a haemocytometer at days indicated. MTS and SYBR-DNA assays were performed, as detailed in materials and methods, at days indicated. The treatment results are shown as a percentage of the uninduced vehicle control (±SE) correlating with viable cell number (Trypan blue counts), colorimetric measurement (MTS), and fluorescent intensity (SYBR assay). Each experiment was performed in triplicate at least 3 times with similar results.

Mentions: In this study we analyzed the anti-proliferative effects of the pure anti-estrogen ICI 182780, p14ARF-p53 induction, FTY720 drug treatment and serum starvation in MCF-7 cells. These experiments were conducted with MCF-7 cells showing p14ARF induction with IPTG. Without IPTG induction, these MCF-7 cells retained all the characteristics of native breast cancer cells including their responsiveness to anti-estrogen treatment (unpublished data). We compared viability of cells post-treatment using the Trypan Blue exclusion assay measured by direct cell counts, the MTS assay by colorimetric measurement, and SYBR-DNA assay measured by cell fluorescence. For simplicity we expressed the results as a mean percentage of control ± SE to allow for direct comparison of all assays (Fig. 1).


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)

Comparative analysis of MCF-7 cell viability, cell number and mitochondrial activity.Cells were treated with 5 mM IPTG, 10 nM ICI 182780, 5 µM FTY720, or serum deprived (serum free) 48 h post-seeding. Using the Trypan Blue exclusion method cells were harvested and viable cells counted using a haemocytometer at days indicated. MTS and SYBR-DNA assays were performed, as detailed in materials and methods, at days indicated. The treatment results are shown as a percentage of the uninduced vehicle control (±SE) correlating with viable cell number (Trypan blue counts), colorimetric measurement (MTS), and fluorescent intensity (SYBR assay). Each experiment was performed in triplicate at least 3 times with similar results.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020623-g001: Comparative analysis of MCF-7 cell viability, cell number and mitochondrial activity.Cells were treated with 5 mM IPTG, 10 nM ICI 182780, 5 µM FTY720, or serum deprived (serum free) 48 h post-seeding. Using the Trypan Blue exclusion method cells were harvested and viable cells counted using a haemocytometer at days indicated. MTS and SYBR-DNA assays were performed, as detailed in materials and methods, at days indicated. The treatment results are shown as a percentage of the uninduced vehicle control (±SE) correlating with viable cell number (Trypan blue counts), colorimetric measurement (MTS), and fluorescent intensity (SYBR assay). Each experiment was performed in triplicate at least 3 times with similar results.
Mentions: In this study we analyzed the anti-proliferative effects of the pure anti-estrogen ICI 182780, p14ARF-p53 induction, FTY720 drug treatment and serum starvation in MCF-7 cells. These experiments were conducted with MCF-7 cells showing p14ARF induction with IPTG. Without IPTG induction, these MCF-7 cells retained all the characteristics of native breast cancer cells including their responsiveness to anti-estrogen treatment (unpublished data). We compared viability of cells post-treatment using the Trypan Blue exclusion assay measured by direct cell counts, the MTS assay by colorimetric measurement, and SYBR-DNA assay measured by cell fluorescence. For simplicity we expressed the results as a mean percentage of control ± SE to allow for direct comparison of all assays (Fig. 1).

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