A Simple High-Content Cell Cycle Assay Reveals Frequent Discrepancies between Cell Number and ATP and MTS Proliferation Assays

PLoS ONE

Volumn 8, Issue 5, 2013, Pages

  Chan, Grace Ka Yan ^a   Kleinheinz, Tracy L ^a   Peterson, David ^a   Moffat, John G ^a  

^a GENENTECH INC   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

3 (4,5 DIMETHYLTHIAZOL 2 YL) 5 (3 CARBOXYMETHOXYPHENYL) 2 (4 SULFOPHENYL) 2H TETRAZOLIUM; 4 (8 CYCLOPENTYL 7 ETHYL 5,6,7,8 TETRAHYDRO 5 METHYL 6 OXO 2 PTERIDINYLAMINO) 3 METHOXY N (1 METHYL 4 PIPERIDINYL)BENZAMIDE; ADENOSINE TRIPHOSPHATE; APHIDICOLIN; BREFELDIN A; CISPLATIN; COLCHICINE; CRIZOTINIB; CYTOCHALASIN D; DACTINOMYCIN; DEMECOLCINE; DOXORUBICIN; EPOTHILONE B; ETOPOSIDE; FLUORESCENT DYE; FLUOROURACIL; GEMCITABINE; N [5 (5 TERT BUTYL 2 OXAZOLYLMETHYLTHIO) 2 THIAZOLYL]ISONIPECOTAMIDE; NOCODAZOLE; PACLITAXEL; PURVALANOL A; STAUROSPORINE; TETRAZOLIUM; TIVANTINIB; TOZASERTIB; TRICHOSTATIN A; UNCLASSIFIED DRUG; VINBLASTINE; VINCRISTINE; ANTINEOPLASTIC AGENT;

ANTIPROLIFERATIVE ACTIVITY; ARTICLE; BIOLUMINESCENCE; CELL ASSAY; CELL COUNT; CELL CYCLE ARREST; CELL CYCLE PARAMETERS; CELL CYCLE PHASE; CELL LINE; CELL METABOLISM; CELL PROLIFERATION; CELL SIZE; CELL VIABILITY; DNA BINDING; DNA SYNTHESIS; DRUG EFFICACY; DRUG MECHANISM; DRUG POTENCY; ENZYME INHIBITION; HUMAN; HUMAN CELL; MITOCHONDRION; MOLECULAR IMAGING; PHENOTYPE; CELL CYCLE; DOSE RESPONSE; DRUG DEVELOPMENT; DRUG EFFECTS; EVALUATION STUDY; HIGH THROUGHPUT SCREENING; LUMINESCENCE; PHYSIOLOGY; PROCEDURES; TUMOR CELL LINE;

ADENOSINE TRIPHOSPHATE; ANTINEOPLASTIC AGENTS; CELL COUNT; CELL CYCLE; CELL LINE, TUMOR; CELL PROLIFERATION; DOSE-RESPONSE RELATIONSHIP, DRUG; DRUG DISCOVERY; HIGH-THROUGHPUT SCREENING ASSAYS; HUMANS; LUMINESCENT MEASUREMENTS; TETRAZOLIUM SALTS;

EID: 84877856607     PISSN: None     EISSN: 19326203     Source Type: Journal    
DOI: 10.1371/journal.pone.0063583     Document Type: Article

References (47)

1. Quent VMC, Loessner D, Friis T, Reichert JC, Hutmacher DW, (2010) Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research. J Cell Mol Med 14: 1003-1013 doi:10.1111/j.1582-4934.2010.01013.x.
2. Crouch SP, Kozlowski R, Slater KJ, Fletcher J, (1993) The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. J Immunol Methods 160: 81-88.
3. Mueller H, Kassack MU, Wiese M, (2004) Comparison of the usefulness of the MTT, ATP, and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines. J Biomol Screen 9: 506-515 doi:10.1177/1087057104265386.
4. Hannah R, Beck M (2001) Celltiter-Glo™ Luminescent Cell Viability Assay: A Sensitive And Rapid Method For Determining Cell Viability. Promega Cell Notes.
5. Berridge M, Herst P, Tan A, (2005) Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction. BIOTECHNOLOGY ANNUAL REVIEW 11: 127-152 doi:10.1016/S1387-2656(05)11004-7.
6. Denizot F, Lang R, (1986) Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89: 271-277.
7. Loveland BE, Johns TG, Mackay IR, Vaillant F, Wang ZX, et al. (1992) Validation of the MTT dye assay for enumeration of cells in proliferative and antiproliferative assays. Biochem Int 27: 501-510.
8. Jones LJ, Gray M, Yue ST, Haugland RP, Singer VL, (2001) Sensitive determination of cell number using the CyQUANT cell proliferation assay. J Immunol Methods 254: 85-98.
9. Fu X, Wan S, Lyu YL, Liu LF, Qi H, (2008) Etoposide induces ATM-dependent mitochondrial biogenesis through AMPK activation. PLoS ONE 3: e2009 doi:10.1371/journal.pone.0002009.
10. Kluza J, Marchetti P, Gallego MA, Lancel S, Fournier C, et al. (2004) Mitochondrial proliferation during apoptosis induced by anticancer agents: effects of doxorubicin and mitoxantrone on cancer and cardiac cells. Oncogene 23: 7018-7030 doi:10.1038/sj.onc.1207936.
11. Maldonado EN, Patnaik J, Mullins MR, Lemasters JJ, (2010) Free tubulin modulates mitochondrial membrane potential in cancer cells. Cancer Res 70: 10192-10201 doi:10.1158/0008-5472.CAN-10-2429.
12. Oropesa M, la Mata de M, Maraver JG, Cordero MD, Cotán D, et al. (2011) Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria. Apoptosis 16: 404-424 doi:10.1007/s10495-011-0577-1.
13. Reipert S, Berry J, Hughes MF, Hickman JA, Allen TD, (1995) Changes of mitochondrial mass in the hemopoietic stem cell line FDCP-mix after treatment with etoposide: a correlative study by multiparameter flow cytometry and confocal and electron microscopy. Exp Cell Res 221: 281-288 doi:10.1006/excr.1995.1376.
14. Renner K, Amberger A, Konwalinka G, Kofler R, Gnaiger E, (2003) Changes of mitochondrial respiration, mitochondrial content and cell size after induction of apoptosis in leukemia cells. Biochim Biophys Acta 1642: 115-123.
15. Lundholt BK, Scudder KM, Pagliaro L, (2003) A simple technique for reducing edge effect in cell-based assays. J Biomol Screen 8: 566-570 doi:10.1177/1087057103256465.
16. Solit DB, Garraway LA, Pratilas CA, Sawai A, Getz G, et al. (2006) BRAF mutation predicts sensitivity to MEK inhibition. Nature 439: 358-362 doi:10.1038/nature04304.
17. Gysin S, Lee SH, Dean NM, McMahon M, (2005) Pharmacologic inhibition of RAF->MEK->ERK signaling elicits pancreatic cancer cell cycle arrest through induced expression of p27Kip1. Cancer Res 65: 4870-4880 doi:10.1158/0008-5472.CAN-04-2848.
18. Harrington EA, Bebbington D, Moore J, Rasmussen RK, Ajose-Adeogun AO, et al. (2004) VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nature medicine 10: 262-267 doi:10.1038/nm1003.
19. Cui JJ, Tran-Dubé M, Shen H, Nambu M, Kung PP, et al. (2011) Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK). J Med Chem 54: 6342-6363 doi:10.1021/jm2007613.
20. Steegmaier M, Hoffmann M, Baum A, Lénárt P, Petronczki M, et al. (2007) BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo. Curr Biol 17: 316-322 doi:10.1016/j.cub.2006.12.037.
21. Lénárt P, Petronczki M, Steegmaier M, Di Fiore B, Lipp JJ, et al. (2007) The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1. Curr Biol 17: 304-315 doi:10.1016/j.cub.2006.12.046.
22. Sutherland JJ, Low J, Blosser W, Dowless M, Engler TA, et al. (2011) A robust high-content imaging approach for probing the mechanism of action and phenotypic outcomes of cell-cycle modulators. Mol Cancer Ther 10: 242-254 doi:10.1158/1535-7163.MCT-10-0720.
23. Munshi N, Jeay S, Li Y, Chen CR, France DS, et al. (2010) ARQ 197, a Novel and Selective Inhibitor of the Human c-Met Receptor Tyrosine Kinase with Antitumor Activity. Mol Cancer Ther 9: 1544-1553 doi:10.1158/1535-7163.MCT-09-1173.
24. Basilico C, Pennacchietti S, Vigna E, Chiriaco C, Arena S, et al. (2013) Tivantinib (ARQ197) displays cytotoxic activity that is independent of its ability to bind MET. Clin Cancer Res. doi:10.1158/1078-0432.CCR-12-3459.
25. Cottet-Rousselle C, Ronot X, Leverve X, Mayol JF, (2011) Cytometric assessment of mitochondria using fluorescent probes. Cytometry A 79: 405-425 doi:10.1002/cyto.a.21061.
26. Johnson I, editor (2011) Probes for Mitochondria- section 12.2. The Molecular Probes Handbook- A guide to fluorescent probes and labeling technologies.
27. Posakony JW, England JM, Attardi G, (1977) Mitochondrial growth and division during the cell cycle in HeLa cells. J Cell Biol 74: 468-491.
28. Lyman SK, Crawley SC, Gong R, Adamkewicz JI, McGrath G, et al. (2011) High-content, high-throughput analysis of cell cycle perturbations induced by the HSP90 inhibitor XL888. PLoS ONE 6: e17692 doi:10.1371/journal.pone.0017692.
29. Neumann B, Walter T, Hériché JK, Bulkescher J, Erfle H, et al. (2010) Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes. Nature 464: 721-727 doi:10.1038/nature08869.
30. Feng Y, Mitchison TJ, Bender A, Young DW, Tallarico JA, (2009) Multi-parameter phenotypic profiling: using cellular effects to characterize small-molecule compounds. Nat Rev Drug Discov 8: 567-578 doi:10.1038/nrd2876.
31. Loo LH, Lin HJ, Steininger RJ, Wang Y, Wu LF, et al. (2009) An approach for extensibly profiling the molecular states of cellular subpopulations. Nat Methods 6: 759-765 doi:10.1038/nmeth.1375.
32. Perlman ZE, Slack MD, Feng Y, Mitchison TJ, Wu LF, et al. (2004) Multidimensional drug profiling by automated microscopy. Science 306: 1194-1198 doi:10.1126/science.1100709.
33. Newman RH, Zhang J, (2008) Fucci: street lights on the road to mitosis. Chem Biol 15: 97-98 doi:10.1016/j.chembiol.2008.02.003.
34. Gasparri F, Ciavolella A, Galvani A, (2007) Cell-cycle inhibitor profiling by high-content analysis. Advances in experimental medicine and biology, Vol. 604: 137-148 doi:10.1007/978-0-387-69116-9-13.
35. Heng BC, Das GK, Zhao X, Ma LL, Tan TTY, et al. (2010) Comparative cytotoxicity evaluation of lanthanide nanomaterials on mouse and human cell lines with metabolic and DNA-quantification assays. Biointerphases 5: FA88-FA97 doi:10.1116/1.3494617.
36. Wang P, Henning SM, Heber D, (2010) Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols. PLoS ONE 5: e10202 doi:10.1371/journal.pone.0010202.
37. Sakaue-Sawano A, Kobayashi T, Ohtawa K, Miyawaki A, (2011) Drug-induced cell cycle modulation leading to cell-cycle arrest, nuclear mis-segregation, or endoreplication. BMC Cell Biol 12: 2 doi:10.1186/1471-2121-12-2.
38. Montecucco A, Biamonti G, (2007) Cellular response to etoposide treatment. Cancer Lett 252: 9-18 doi:10.1016/j.canlet.2006.11.005.
39. Sims JT, Plattner R, (2009) MTT assays cannot be utilized to study the effects of STI571/Gleevec on the viability of solid tumor cell lines. Cancer Chemother Pharmacol 64: 629-633 doi:10.1007/s00280-009-1004-y.
40. Pagliacci MC, Spinozzi F, Migliorati G, Fumi G, Smacchia M, et al. (1993) Genistein inhibits tumour cell growth in vitro but enhances mitochondrial reduction of tetrazolium salts: a further pitfall in the use of the MTT assay for evaluating cell growth and survival. Eur J Cancer 29A: 1573-1577.
41. McGowan EM, Alling N, Jackson EA, Yagoub D, Haass NK, et al. (2011) Evaluation of cell cycle arrest in estrogen responsive MCF-7 breast cancer cells: pitfalls of the MTS assay. PLoS ONE 6: e20623 doi:10.1371/journal.pone.0020623.
42. Rudolf K, Cervinka M, Rudolf E, (2009) Cytotoxicity and mitochondrial apoptosis induced by etoposide in melanoma cells. Cancer Invest 27: 704-717 doi:10.1080/07357900802653480.
43. Kitami T, Logan DJ, Negri J, Hasaka T, Tolliday NJ, et al. (2012) A chemical screen probing the relationship between mitochondrial content and cell size. PLoS ONE 7: e33755 doi:10.1371/journal.pone.0033755.
44. Wagner BK, Kitami T, Gilbert TJ, Peck D, Ramanathan A, et al. (2008) Large-scale chemical dissection of mitochondrial function. Nat Biotechnol 26: 343-351 doi:10.1038/nbt1387.
45. Brand MD, Nicholls DG, (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435: 297-312 doi:10.1042/BJ20110162.
46. Kushnareva Y, Newmeyer DD, (2010) Bioenergetics and cell death. Ann N Y Acad Sci 1201: 50-57 doi:10.1111/j.1749-6632.2010.05633.x.
47. Buchakjian MR, Kornbluth S, (2010) The engine driving the ship: metabolic steering of cell proliferation and death. Nat Rev Mol Cell Biol 11: 715-727 doi:10.1038/nrm2972.