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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)

Abstract

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

 

Seed(cells/mL/well)

Refeed(after seeding)

Incubation with[3H]E2

Endpoint

Measurement

MCF-7 Focus

1 × 105

24 h and every

None

Development of

Increase or

assay

 

3-4 d for a

 

foci

decrease in focal

  

total of 4

 

Inhibition of focus

retention of

  

refeeds

 

development

rhodamine B stain

Whole-cell

5 × 105

24 h

For 3-4 h; at the

Displacement of

Decrease of [3H]E2

ER-binding

  

same time as test

[3H]E2 from ER

in cells, with

assay

  

agent or a specified

by test agent

increase in test

   

time after test agent;

 

agent

   

at either 4 or 37°C

  

Radiometric

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

[3H]E2

  

series of time points;

in production of

in test agent

   

at 37†C

Tritiated H2O

 

Keywords

Test Agent Tissue Culture Medium Automate Cell Counter Bovine Calf Serum Human Breast Adenocarcinoma 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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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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