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

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.

	Seed(cells/mL/well)	Refeed(after seeding)	Incubation with[3 H]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 [3 H]E2
ER-binding			same time as test	[3 H]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	H2 O in media,
catabolism of			test agent for a	[3 H]E2 , resulting	with increase
[3 H]E2			series of time points;	in production of	in test agent
			at 37†C	Tritiated H2 O