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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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Flow cytometric analysis of cell cycle phases post ICI 182780 and IPTG treatment.Cells were treated with 10 nM ICI 182780 or 5 mM IPTG (p14ARF-induction) 48 h post seeding. At 48 h post-treatment cells were harvested, stained with propidium iodide solution as described in materials and methods and analysed for cell cycle distribution by flow cytometry using Modfit software. A. Fluorescence histograms showing cell cycle distribution of control, IPTG and ICI 182780 treated MCF-7 cells (representative experiment). B. Representative column graph showing the percentage of cells (± SD) in each cell cycle. This experiment was performed three times with three different cell lines showed similar results.
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pone-0020623-g002: Flow cytometric analysis of cell cycle phases post ICI 182780 and IPTG treatment.Cells were treated with 10 nM ICI 182780 or 5 mM IPTG (p14ARF-induction) 48 h post seeding. At 48 h post-treatment cells were harvested, stained with propidium iodide solution as described in materials and methods and analysed for cell cycle distribution by flow cytometry using Modfit software. A. Fluorescence histograms showing cell cycle distribution of control, IPTG and ICI 182780 treated MCF-7 cells (representative experiment). B. Representative column graph showing the percentage of cells (± SD) in each cell cycle. This experiment was performed three times with three different cell lines showed similar results.

Mentions: The above observations showed distinct discrepancies between the MTS and SYBR-DNA assay readouts and cell counts when cells were treated with ICI 182780 and IPTG (induction of p14ARF) at days 1 and 2. Flow cytometric analysis confirmed cell cycle inhibition post treatment with ICI 182780 or IPTG (Fig. 2), concordant with the cell count results (Fig. 1). Flow cytometric results presented additional information on cell behaviour post drug treatment; IPTG induction of p14ARF significantly reduced the S-phase peak with an accumulation of cells in the G1 and G2/M cell-cycle phases whereas ICI 182780-treated cells accumulated in the G1/GO phase (Fig. 2A). These experiments were repeated with three different p14ARF-clonal cell lines with similar results to ensure that G2/M accumulation post-IPTG treatment was not due to aberrant clonal variation (Fig. 2B). Our results are consistent with the published literature, which demonstrate ICI 182780 induces quiescence (G1/G0) in MCF-7cells [2], and p14ARF-p53 arrests cells in both G1 and G2/M cell-cycle phases [19].


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)

Flow cytometric analysis of cell cycle phases post ICI 182780 and IPTG treatment.Cells were treated with 10 nM ICI 182780 or 5 mM IPTG (p14ARF-induction) 48 h post seeding. At 48 h post-treatment cells were harvested, stained with propidium iodide solution as described in materials and methods and analysed for cell cycle distribution by flow cytometry using Modfit software. A. Fluorescence histograms showing cell cycle distribution of control, IPTG and ICI 182780 treated MCF-7 cells (representative experiment). B. Representative column graph showing the percentage of cells (± SD) in each cell cycle. This experiment was performed three times with three different cell lines showed similar results.
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Related In: Results  -  Collection

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

pone-0020623-g002: Flow cytometric analysis of cell cycle phases post ICI 182780 and IPTG treatment.Cells were treated with 10 nM ICI 182780 or 5 mM IPTG (p14ARF-induction) 48 h post seeding. At 48 h post-treatment cells were harvested, stained with propidium iodide solution as described in materials and methods and analysed for cell cycle distribution by flow cytometry using Modfit software. A. Fluorescence histograms showing cell cycle distribution of control, IPTG and ICI 182780 treated MCF-7 cells (representative experiment). B. Representative column graph showing the percentage of cells (± SD) in each cell cycle. This experiment was performed three times with three different cell lines showed similar results.
Mentions: The above observations showed distinct discrepancies between the MTS and SYBR-DNA assay readouts and cell counts when cells were treated with ICI 182780 and IPTG (induction of p14ARF) at days 1 and 2. Flow cytometric analysis confirmed cell cycle inhibition post treatment with ICI 182780 or IPTG (Fig. 2), concordant with the cell count results (Fig. 1). Flow cytometric results presented additional information on cell behaviour post drug treatment; IPTG induction of p14ARF significantly reduced the S-phase peak with an accumulation of cells in the G1 and G2/M cell-cycle phases whereas ICI 182780-treated cells accumulated in the G1/GO phase (Fig. 2A). These experiments were repeated with three different p14ARF-clonal cell lines with similar results to ensure that G2/M accumulation post-IPTG treatment was not due to aberrant clonal variation (Fig. 2B). Our results are consistent with the published literature, which demonstrate ICI 182780 induces quiescence (G1/G0) in MCF-7cells [2], and p14ARF-p53 arrests cells in both G1 and G2/M cell-cycle phases [19].

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