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.