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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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Induction of p14ARF increases mitochondrial biomass and maintains membrane potential.A. 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. Images processed by high content imaging (magnification ×200) and mitochondria (per cell) counted using BD Attovision™ software. C. 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 ΔΨmt in IPTG-treated cells. D. 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.
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pone-0020623-g005: Induction of p14ARF increases mitochondrial biomass and maintains membrane potential.A. 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. Images processed by high content imaging (magnification ×200) and mitochondria (per cell) counted using BD Attovision™ software. C. 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 ΔΨmt in IPTG-treated cells. D. 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.

Mentions: To understand the mechanism underlying the increased metabolic activity in MCF-7 cells with p14ARF induction, mitochondrial content and the mitochondrial membrane potential in treated and untreated cells were analysed. At day 3 post-IPTG-treatment, MitoTracker™ red, CellTracker™ green, and Hoechst 33342 was added to the cell cultures and live cells were imaged (Fig. 5A). A noticeable increase in cell size by day 3 was observed concomitant with an increase in mitochondria density per cell; this increase in cell size is consistent with the senescence-phenotype. Mitochondria cell number was quantified using the BD pathway Bioimager and Attovision software (Fig. 5B). A 3-fold increase (P<0.001) in mitochondrial counts post-p14ARF induction compared to control cells was observed.


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)

Induction of p14ARF increases mitochondrial biomass and maintains membrane potential.A. 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. Images processed by high content imaging (magnification ×200) and mitochondria (per cell) counted using BD Attovision™ software. C. 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 ΔΨmt in IPTG-treated cells. D. 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020623-g005: Induction of p14ARF increases mitochondrial biomass and maintains membrane potential.A. 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. Images processed by high content imaging (magnification ×200) and mitochondria (per cell) counted using BD Attovision™ software. C. 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 ΔΨmt in IPTG-treated cells. D. 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.
Mentions: To understand the mechanism underlying the increased metabolic activity in MCF-7 cells with p14ARF induction, mitochondrial content and the mitochondrial membrane potential in treated and untreated cells were analysed. At day 3 post-IPTG-treatment, MitoTracker™ red, CellTracker™ green, and Hoechst 33342 was added to the cell cultures and live cells were imaged (Fig. 5A). A noticeable increase in cell size by day 3 was observed concomitant with an increase in mitochondria density per cell; this increase in cell size is consistent with the senescence-phenotype. Mitochondria cell number was quantified using the BD pathway Bioimager and Attovision software (Fig. 5B). A 3-fold increase (P<0.001) in mitochondrial counts post-p14ARF induction compared to control cells was observed.

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