Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research

Abstract

Cell proliferation is a critical and frequently studied feature of molecular biology in cancer research. Therefore, various assays are available using different strategies to measure cell proliferation. Metabolic assays such as AlamarBlue, water-soluble tetrazolium salt and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, which were originally developed to determine cell toxicity, are used to assess cell numbers. Additionally, proliferative activity can be determined by quantification of DNA content using fluorophores such as CyQuant and PicoGreen. Referring to data published in high ranking cancer journals, these assays were applied in 945 publications over the past 14 years to examine the proliferative behaviour of diverse cell types. In these studies, however, mainly metabolic assays were used to quantify changes in cell growth yet these assays may not accurately reflect cellular proliferation rates due to a miscorrelation of metabolic activity and cell number. Testing this hypothesis, we compared the metabolic activity of different cell types, human cancer cells and primary cells, over a time period of 4 days using AlamarBlue and the fluorometric assays CyQuant and PicoGreen to determine their DNA content. Our results show certain discrepancies in terms of over-estimation of cell proliferation with respect to the metabolic assay in comparison to DNA binding fluorophores.

Fig 1

Number of assays used to assess cell proliferation in publications of high ranking cancer journals; publications were counted from January 1995 to July 2009. A total of 60% of all researchers applied the listed assays to measure cell proliferation. Values for MTT/MTS were assessed through reading the abstracts of Medline articles retrieved through a ‘Google Scholar’ search. In the groups of WST-1, AlamarBlue, PicoGreen and CyQuant every publication was checked individually for the application of those assays.

Fig 2

Light microscopical images of four cell types at different time-points. After 4 h of seeding 8300 cells/cm² OV-MZ-6 and SKOV-3 cells are attached to the bottom of the cell culture plate; bmMSC are not fully attached yet as elongated fibroblast-like features are missing; HUCPVC show fibroblast-like morphology. After 96 h OV-MZ-6 and SKOV-3 cells proliferated and displayed a typical epithelial cobblestone formation; bmMSC showed slight proliferation and fibroblast-like features; HUCPVC showed typical morphological features (magnification 10×, scale bar 100 μm).

Fig 3

Direct comparison of n-fold change in DNA content measured by CyQuant (A) and PicoGreen (B); n-fold change of cell numbers based on metabolic activity assessed by AlamarBlue (C) after 24, 48, 72, 96 hrs of OV-MZ-6 (•), SKOV-3 (▪), HUCPVC (○), bmMSC (X). CyQuant and PicoGreen showed a 7.5/7.2- and 5.3/6.6-fold increase of DNA content of both cancer cell lines SKOV-3 and OV-MZ-6, respectively, between 24 and 96 hrs. However, AlamarBlue estimated a 9.2/9.7-fold-change of cell numbers based on metabolic activity. CyQuant and PicoGreen showed a 2.3/1.1-fold increase of DNA content of HUCPVC and bmMSC, respectively, during the 4-day time span. AlamarBlue estimated a 4.5/1.5-fold change in cell numbers of HUCPVC and bmMSC based on metabolic activity during 24 to 96 hrs in cell culture.

Fig 4

Correlations between AlamarBlue/PicoGreen (A) and AlamarBlue/ CyQuant (B) PicoGreen/CyQuant (C) assessing cell proliferation of OV-MZ-6 (•), SKOV-3 (▪), HUCPVC (○), bmMSC (X) based on the results shown in Fig. 3. The estimation of cell proliferation based on AlamarBlue correlated well with 0.7 < R² < 1 with increased DNA measured by CyQuant and PicoGreen except for bmMSC (R²= 0.27/0.32). However, this technique resulted in an over-estimation of cell proliferation from 21% to 64%. PicoGreen and CyQuant correlated well with 0.82 < R² < 0.99 and a deviation from the reference line from 2% to 13%.