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 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

Comparative analysis of MCF-7 cell viability, cell number and mitochondrial activity.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020623#s4" target="_blank">materials and methods</a>, 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.</p

EdU incorporation post ICI 182780 and IPTG (p14ARF) treatment.

<p>Cells were seeded on cover slips and treated with 5 mM IPTG or 10 nM ICI 182780 24 h post-seeding. EdU was added to the medium on days 1 and 4 and cells were incubated for a further 20 h. EdU incorporation was visualized by staining with Alexafluor 488 (green). The nucleus was stained with Hoechst 33342 (blue) and images were taken on a Nikon fluorescence microscope (magnification ×200). Column graph shows % cells staining for EdU compared to Hoechst 33342 stained nuclei (± SE). Experiments were performed in triplicate (duplicate biological experiments). A minimum of 500 cells was counted for each treatment.</p

Flow cytometric analysis of cell cycle phases post ICI 182780 and IPTG treatment.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020623#s4" target="_blank">materials and methods</a> 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.</p

ICI 182780 and p14ARF increase mitochondria activity.

<p>Cells were treated with 10 nM ICI 182780 (<b>A</b>) or 5 mM IPTG (<b>B</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.</p

Induction of p14ARF increases mitochondrial biomass and maintains membrane potential.

<p><b>A.</b> Cells were treated with 5 mM IPTG 48 h post-seeding. At day 3 post-IPTG treatment, live cells were incubated with Mitotracker (red), CellTracker (green) and Hoechst 33342 (blue) and imaged using an inverted fluorescent microscope (magnification ×400). Cells treated with IPTG noticeably increased in size. <b>B.</b> Images processed by high content imaging (magnification ×200) and mitochondria (per cell) counted using BD Attovision™ software. <b>C.</b> Cells were treated with 5 mM IPTG 48 h post-seeding. On day 3 post-IPTG-treatment cells were stained with TMRE for 15 min (+), or left unstained (−) and run through a flow cytometer (IPTG = black, control = white). TMRE-IPTG-treated cells showed increased fluorescence intensity compared to the TMRE-control cells, which is indicative of an increase in ΔΨ_mt in IPTG-treated cells. <b>D.</b> The median FL2 relative fluorescence units (RFU) of control and IPTG treated cells (day 3) were determined by flow cytometry. The column graph shows the median RFU of TMRE-stained cells minus unstained cells (±SE). This experiment was performed at least twice in triplicate.</p