Assessing Modulation of Estrogenic Activity of Environmental and Pharmaceutical Compounds Using MCF-7 Focus Assay

  • Kathleen F. Arcaro
  • John F. Gierthy
Part of the Methods in Molecular Biology™ book series (MIMB, volume 176)


The MCF-7 cell line was isolated from a pleural metastasis of a human breast adenocarcinoma, and, when grown on plastic substrates, typically forms a continuous cell monolayer at confluence (1). MCF-7 cell cultures respond to 17β-estradiol (E2) by increases in the expression of a number of genes ((2),(3) and localized focal postconfluent cell proliferation, which results in development of multicellular, three dimensional nodules termed “foci” (4). Thus, focus development in MCF-7 cells may represent the basic characteristics of an estrogenic response, i.e., induction of concerted gene expression, resulting in tissue restructuring through enhanced postconfluent cell proliferation. Since foci are easily counted, the development E2-induced foci and their inhibition are useful as a relevant human-tissue-based assay for the assessment of estrogenic and antiestrogenic activity of environmental and pharmaceutical compounds (5-7). Here the authors give the protocol for measuring focus formation in response to estrogen-modulating agents. In addition, protocols are presented to determine whether the modulation of foci by a particular agent is a result of estrogen-receptor (ER)-dependent activity or changes in the level of E2 through alteration of E2 catabolism. Table 1 provides an overview of the three protocols.
Table 1

Overview of Protocols for Assays a in cpm



Refeed(after seeding)

Incubation with[3H]E2



MCF-7 Focus

1 × 105

24 h and every


Development of

Increase or



3-4 d for a



decrease in focal


total of 4


Inhibition of focus

retention of





rhodamine B stain


5 × 105

24 h

For 3-4 h; at the

Displacement of

Decrease of [3H]E2



same time as test

[3H]E2 from ER

in cells, with



agent or a specified

by test agent

increase in test


time after test agent;




at either 4 or 37°C



5 × 105

24 h

Between 3 and 24 h;

Increase in tritiated

Increase in tritiated

analysis of


after incubation with

catabolism of

H2O in media,

catabolism of


test agent for a

[3H]E2, resulting

with increase



series of time points;

in production of

in test agent


at 37†C

Tritiated H2O



Test Agent Tissue Culture Medium Automate Cell Counter Bovine Calf Serum Human Breast Adenocarcinoma 
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  1. 1.
    Arnold, W. J., Soule, H. D., and Russo, J. (1975) Fine structure of a human mammary carcinoma cell line. In Vitro 10, 356A.Google Scholar
  2. 2.
    Pourreau-Schneider, N., Berthios, Y., Mittre H., Charpin C., Jacquemier J., and Martin P. M. (1984) Estrogen response of MCF-7 cells grown on diverse substrates and in suspension culture: promotion of morphological heterogeneity, modulation of progestin receptor induction; cell-substrate interactions on collagen gels. J. Steroid Biochem. 21, 763–771.PubMedCrossRefGoogle Scholar
  3. 3.
    Dickerman, H. W., Martinez, H. L., Seeger, J. I., and Kumar, S. A. (1989) Estrogen regulation of human breast cancer cell line MCF-7 tissue plasminogen activator. Endocrinology 125, 492–500.PubMedCrossRefGoogle Scholar
  4. 4.
    Gierthy, J. F., Lincoln, D. W. I., Roth, K. E., Bowser, S. S., Bennett, J. A., Bradley, L., and Dickerman, H. W. (1991) Estrogen-stimulation of postconfluent cell accumulation and foci formation of human MCF-7 breast cancer cells. J. Cell. Biochem. 45, 177–187.PubMedCrossRefGoogle Scholar
  5. 5.
    Arcaro, K. F., Yi, L. D., Seegal, R. F., Vakharia, D. D., Yang, Y., Spink, D. C., Brosch, K., and Gierthy, J. F. (1999) 2,2′,6,6′-tetrachlorobiphenyl is estrogenic in vitro and in vivo. J. Cell. Biochem. 72, 94–102.PubMedCrossRefGoogle Scholar
  6. 6.
    Arcaro, K. F., O'Keefe, P. W., Yang, Y., Clayton, W., and Gierthy, J. F. (1999) Antiestrogenicity of environmental polycyclic aromatic hydrocarbons in human breast cancer cells. Toxicology 133, 115–127.PubMedCrossRefGoogle Scholar
  7. 7.
    Arcaro, K. F., Yang, Y., Vakharia, D. D., and Gierthy, J. F. (2000) Toxaphene is antiestrogenic in a human breast-cancer cell assay. J. Toxicol. Environ. Health 59, 101–114.CrossRefGoogle Scholar
  8. 8.
    Soto, A. M., Justicia, H., Wray, J. W., and Sonnenschein, C. (1991) p-Nonylphenol: an estrogenic xenobiotic released from “modified” polystyrene. Environ. Health Perspect. 92, 167–173.PubMedCrossRefGoogle Scholar
  9. 9.
    Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., et al. (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82, 1107–1112.PubMedCrossRefGoogle Scholar
  10. 10.
    Arcaro, K. F., Yang, Y., and Gierthy, J. F. (2000) Benzo[k]fluoranthene enhancement and suppression of 17β-estradiol catabolism in MCF-7 breast cancer cells. J. Toxicol. Environ. Health. 58, 413–426.CrossRefGoogle Scholar
  11. 11.
    Faessel, H. M., Levasseur, L. M., Slocum, H. K., and Greco, W. R.(1999) Parabolic growth patterns in 96-well plate cell growth experiments. In Vitro Cell. Dev. Biol. Animal 35, 270–278.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Kathleen F. Arcaro
    • 1
  • John F. Gierthy
    • 2
  1. 1.Department of Environmental Health and Toxicology, School of Public HealthUniversity at AlbanyRensselaerNY
  2. 2.Wadsworth Center for Laboratories and ResearchNew York State Department of HealthAlbanyNY

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