1 In vitro Assays
1.1 Radiometric Binding Assay
The radiometric binding assay is a standard procedure in many academic and industrial re-search groups to determine the relative binding affinity (RBA) of new substances with poten-tial estrogenic or antiestrogenic activity. It is an indirect method based on the competitive displacement of the tritium labelled physiological ligand 17β-estradiol ([3H]-estradiol) and circumvents the synthesis and application of radioactive test compounds.
Increasing concentrations of inhibitor compete with the tracer molecule [3H]-estradiol, applied in constant concentration, for the single binding site at the ER. The degree of displacement of [3H]-estradiol from the receptor is direct proportional to the relative binding affinity of the competitor.
The origin of receptor material used in this binding assay varies. A natural source of receptor is the cytosol prepared from uteri of immature animals such as calves [Walter et al., 2004], lambs [Stauffer et al., 2001] or rats [Katzenellenbogen et al., 1973; Williams et al., 1974], which possess low levels of endogenous estrogen. This cytosol contains both estrogen recep-tor subtypes ERα and ERβ, but the predominantly expressed receprecep-tor in the uterus is ERα [Enmark and Gustafsson, 1998].
Full-length, human receptors ERα and ERβ expressed as recombinant proteins in baculovirus infected insect cells are also used for this purpose. With these proteins it was possible to es-tablish a new binding assay in our research group to assign the affinity and selectivity of cer-tain compounds to one or the other ER isoform.
Depending on the receptor source different work-up procedures are required to separate the excess of radioactivity. In case of the receptor containing cytosol unbound [3H]-estradiol is removed by dextran-coated charcoal (DCC), following the recommendation of EORTC [1973]. The pretreatment with dextran (60-90kDa) is necessary to close the large pores of the charcoal and reach effective absorption of excess [3H]-estradiol and other small molecules that are found in the cytosol, but not of the receptor-ligand-complex. The DCC method fails when the recombinant receptors are used. These proteins are substantially smaller than those
obtained from natural sources and are removed together with unbound [3H]-estradiol, so that no receptor-bound radioactivity can be detected. For this reason the receptor-ligand-complex formed during equilibration is absorbed with hydroxylapatite (HAP) and subsequently washed free of any unbound radioactivity. The HAP pellet is resuspended and counted for tritium activity in a liquid scintillation counter [Ke et al., 1998; Leake and Habib, 1987]
For each binding assay carried out with either natural or recombinant receptors control and background values are determined. In the control experiment the maximum number of bind-ing sites is determined by usbind-ing exclusively tritium labelled estradiol as ligand. The back-ground experiment takes into account any low affinity binding sites, such as other lipids or proteins, that especially come along with the preparation of the cytosol and might be respon-sible for unspecific and irreverrespon-sible binding of estradiol or test compounds. For this reason, an excess concentration of the unlabeled estradiol is incubated with [3H]-estradiol and so the reversible equilibrium shifted quantitatively favouring the binding of “cold” ligand to the re-ceptor. Finally, after treatment with DCC or HAP, only unspecific binding is recorded and this background activity is subtracted from all other measurements.
All new compounds and unlabeled estradiol are tested within a broad range of concentrations to identify the molar concentration required to decrease the specific radioligand binding by 50% (IC50-value). The RBA of each competitor is calculated as the ratio of IC50-value of es-tradiol to IC50-value of competitor, multiplied by 100.
1.2 Proliferation Assay with Human Mammary Carcinoma Cell Lines
Since receptor binding affinities do not allow conclusions about the hormonal activity of po-tential antitumour agents, the antiproliferative effect of these substances on human mammary carcinoma cell lines is determined using a computerised microculture chemosensitivity assay based on the quantification of cellmass by staining cells with crystal violet. [Bernhardt et al., 1992]
Two different human breast cancer cell lines have been used. The MCF-7 cell line was estab-lished from a pleural effusion from a disseminated breast carcinoma of a 69 years old patient [Soule et al., 1973]. It is characterised by a high content of estrogen receptors (cf. table B1) and is therefore used to demonstrate an estrogen receptor mediated effect of test compounds.
The MDA-MB-231 cell line is hormone-independent and is derived from the pleural effusion of a 51 years old woman with recidivous adenocarcinoma of the breast [Cailleau et al., 1974].
Its low content of estrogen receptors (cf. table B1) excludes a receptor mediated drug action, but it allows the detection non-specific cytotoxic action of test compounds.
Cell line Passage ERa PRa
MDA-MB-231 32b 3 1
37b 2 7
MCF-7 154b 2 39
166b 119 8
177b 148 5
194c 101 57
198c 280 17
202c 247 1
213c 489 130
Table B1: Steroid receptor content of two human breast cancer cells
aconcentration of receptor in fmol/mg soluble protein
b[Bernhard et al., 1992]
c[Leichtl, 1994]
The ER content of the hormone sensitive MCF-7 cells varies with the number of passages.
Constant culture conditions indicate an increase in ER content with increasing numbers of passages [Leichtl, 1994]. Shortly after rethawing, the ER content is at a limit of detection [Bernhardt et al., 1992]. For this reason, cells are submitted to the chemosensitivity assay not before three or four passages.
In the proliferation assay the MCF-7 cells are stimulated with estradiol in a concentration of 1nM. This resembles the physiological concentration of the hormone in the malignant breast cancer tissue. With this method the chemosensitivity assay gives reproducible results inde-pendent from the passage number and thus indeinde-pendent from the ER content [Walter et al., 2002]. The assay is performed as single-point determination in ctFCS supplemented medium.
The duration of incubation after substance addition is between 200 and 250 hours and the cells are fixed shortly before confluence.
The proliferation assay with the hormone-independent MDA-MB-231 cells is also performed as single-point determination. The concentrations of test compounds are 1µM, 5µM and
10µM and incubation lasts 90 to 100 hours. Since these cells lack the estrogen receptor, estra-diol was omitted and untreated FCS was added to the medium (cf. section E3.2.5).
Each compound is tested in duplicate or triplicate in two or three independent experiments.
Fulvestrant and 4-hydroxytamoxifen are used as references. In order to distinguish inhibitory effects from cytocidal drug action T/C-values are corrected for the initial cell density, which refer to the average cellmass at the time of drug addition.
1.3 Luciferase Assay
The luciferase assay is a convenient, rapid and very sensitive method for the determination of the hormonal activity of test compounds in vitro. The assay is based on the transfection of an suitable eukaryotic cell line with an ERE controlled luciferase reporter gene, that was isolated from the North American firefly Photinus pyralis [de Wet et al., 1987].