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

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 1Overview of Protocols for Assaysain cpm

Seed(cells/mL/well)

Refeed(after seeding)

Incubation with[3 H]E2

Endpoint

Measurement

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