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GFP-reporter for a high throughput assay to monitor estrogenic compounds
J. Biochem. Biophys. Methods 64 (2005) 19 – 37 www.elsevier.com/locate/jbbm
GFP-reporter for a high throughput assay to monitor estrogenic compounds Verena Beck, Angelika Pfitscher, Alois Jungbauer* Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria Received 10 February 2005; received in revised form 21 April 2005; accepted 9 May 2005
Abstract In vitro test systems using yeast cells are a useful tool for the determination of the estrogenic activity of estrogens, phyto- and xeno-estrogens and can be used for monitoring large sample numbers in a routine analysis procedure. Our conventional transactivation assay functions with an expression plasmid expressing estrogen receptor a (ERa) under the control of a copper-inducible CUP1 promoter and a reporter plasmid expressing h-galactosidase under the control of the vitellogenin estrogen response element (ERE). In the novel yeast screen system the lacZ gene in the reporter plasmid was substituted by a gene for green fluorescent protein (GFP). Incubation of yeast with various concentrations of estrogenically active substances led to expression of the reporter gene product GFP in a dose dependent manner. The yeast transactivation assay was further down-scaled to be performed in a microplate scale, which is an important step to facilitate handling of large sample numbers. The sensitivity and reproducibility of the novel test system could be confirmed by analysis of the potencies of various estrogenically active substances. Thus, the newly developed yeast estrogen screen using GFP as a reporter can substitute the assay that has been used for a period of several years. D 2005 Elsevier B.V. All rights reserved. Keywords: 17h-estradiol; Ethinylestradiol; Mestranol; Green fluorescent protein (GFP); Reporter gene; Estrogen receptor a
* Corresponding author. Tel.: +43 1 360066226; fax: +43 1 3697615. E-mail address: [email protected] (A. Jungbauer). 0165-022X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2005.05.001
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1. Introduction The development of rapid and simple in vitro test systems for high-throughput screening for the detection and characterization of substances with estrogenic activity is of interest for the assessment of (a) the potency of newly synthesized drug candidates for treatment of diseases related to estrogens, (b) the potency of plant extracts intended for use as herbal remedies, (c) the potency of metabolites of steroids or polyphenols in urine, feces or plasma and (d) for the analysis of environmental samples and xenoestrogens to perform risk assessment studies. The advantages of in vitro tests are their cost-effectiveness, their high sensitivity and specificity and the high throughput compared to in vivo test systems, although these in vitro systems can never replace in vivo studies. Various in vitro test systems such as receptor binding tests [1–4], cell proliferation assays [1,2,5] or reporter gene assays [6–10] are in use. The latter test system employs the transactivating potential of estrogenically active substances on the hormone receptor [11–13]. Many chemicals exerting estrogenic activity act through the estrogen receptor pathway. The mechanism of action on both known estrogen receptors, estrogen receptor alpha (ERa) and estrogen receptor beta (ERh) [14], is the same, but a substance can have a different transactivational potential on both ERs [4,15,16] which are expressed in different tissues [17,18]. Estrogen receptors belong to the steroid–thyroid hormone receptor superfamily and are located in the nucleus of the cell. In their inactive form they are associated to heat shock proteins in a multiprotein complex [19]. Binding of substances to the ligand binding pocket of the estrogen receptor leads to its activation. The activated, ligandbound estrogen receptor dissociates from the multiprotein complex, dimerizes and moves to the nucleus, where it can bind to an estrogen response element (ERE). The EREs are specific DNA sequences in the promoter region of estrogen target genes. The transcription of these genes is mediated by binding of the activated estrogen receptor dimer to their promoter region. Thus, estrogen receptors act as ligand activated transcription factors. Chemical analyses of estrogenically active substances or binding experiments to the receptor are not always suitable to assess the biological effect of those compounds. Thus, many in vitro reporter gene test systems have been developed that imitate the mechanism of estrogen action via the estrogen receptor pathway and that are suitable to assess the estrogenic potential of a variety of substances. These test systems are either performed in mammalian cell lines expressing the estrogen receptor such as the breast cancer cell line MCF-7 [2,20] or in recombinant yeast strains such as Saccharomyces cerevisiae. Metzger et al. [21] have shown that estrogen receptor functions in yeast. This is the basic finding on which all other yeast reporter systems are based. The test systems developed subsequently either vary in expression plasmid, hormone response element, reporter gene and yeast strain [6,10,11,13,22]. The main advantages of the yeast test systems compared to mammalian cell systems are the cheaper media components and faster growth of yeast, as well as the robustness towards toxic effects such as endotoxins or solvents. With rising sample numbers also the shorter time that is needed for performing the assay is an important factor.
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Many investigators have developed transactivation assays in yeast for their requirements and most of them employ the lacZ gene, which is the gene coding for the enzyme h-galactosidase, as an estrogen-responsive reporter gene [13,22,23]. Concentration of expressed h-galactosidase is correlated to the transactivation potential of the tested compound. Activity of h-galactosidase is detected by photometric measurement of a colored reaction product that develops after cleavage of a colorless substrate [24]. However, for determination of h-galactosidase activity yeast cells have to be disrupted which is a time-consuming procedure. Furthermore cell disruption demands some additional handling steps in the analysis procedure which is a major source of error. An effective possibility to make the reporter gene assay procedure faster is the implementation of green fluorescent protein (GFP) instead of h-galactosidase as a reporter gene. GFP is a protein used very often for various biotechnological studies due to its many application possibilities [25]. The GFP protein is derived from the jelly fish Aequorea victoria and emits green fluorescing light which can be measured directly without cell disintegration [26]. The chromophore of GFP is formed by intramolecular cyclization and subsequent dehydrogenation without adding any cofactors [27]. The green fluorescent protein itself is rather small, highly soluble and stable in a broad pH range [28]. In our lab a yeast transactivation assay for the determination of the estrogenic activity of various samples has been used [8,22,29,30]. The system is based on ubiquitin estrogen receptor fusions yielding into a stably expressed hormone receptor [29]. Subsequent modification of the N-terminus according to the N-end rule from Varshavsky [31] showed effects of receptor expression level and activation function-1 (AF-1) domain on transactivation. This test system has been in use for many years and has been applied for the analysis of various kinds of sample matrices such as the selective estrogen receptor modulators (SERMs) raloxifene [32] and tamoxifen [30], phytoestrogenic preparations [33,34], food samples [35,36] and water samples. In order to simplify the assay procedure the reporter gene lacZ has been replaced by GFP and the performance of both test systems regarding sensitivity and reproducibility was compared. To make the test suited for the analysis of an even larger sample number with smaller volumes it was down-scaled to a microplate scale and it could be shown that results were in agreement with the results obtained in a larger scale.
2. Materials and methods 2.1. Chemicals Buffer reagents, dimethylsulfoxide (DMSO) and o-nitrophenyl-h-galactopyranoside (ONPG) were purchased from Merck (Darmstadt, Germany) or Sigma Aldrich (St. Louis, MO). For yeast media preparation, yeast nitrogen base was obtained from Difco (Franklin Lakes, NJ), amino acids from Serva Feinbiochemica (Heidelberg, Germany) and from Sigma Aldrich (St. Louis, MO). Enzymes for molecular biology were purchased from Fermentas GmbH (St. Leon-Rot, Germany) and electrocompetent E. coli were obtained from Invitrogen (Lofer, Austria). Protease inhibitor cocktail for fungal and yeast cells was
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purchased from Sigma Aldrich (St. Louis, MO). 17h-estradiol, estrone, estriol, 17aestradiol, 17a-ethinylestradiol, diethylstilbestrol, coumestrol, zearalenone and isoflavones were obtained from Sigma Aldrich (St. Louis, MO), 4-nonylphenol (4-NP) technical mixture (PestanalR) was obtained from Riedel-de-Haen, mestranol was purchased from ICN Biomedicals (Eschwege, Germany). Glass beads were purchased from Merck (Darmstadt, Germany). The solvents used were HPLC grade. Black microplates were obtained from Corning (Schiphol-Rijk, The Netherlands), microplates with black walls and transparent bottom were purchased from Perkin Elmer (Boston, MA). 2.2. Construction of plasmids The expression plasmid YEpE12 carrying the human estrogen receptor alpha (ERa), the CUP promoter and a tryptophan–marker and the reporter plasmid YRpE2 (Fig. 1) with two copies of the vitellogenin ERE, the iso-1-cytochrome c (CYC1) promoter in fusion to the lacZ and an uracil auxotrophy marker were a gift from C. R. Lyttle [22]. The expression plasmid YEpE12 was used in the conventional and in the newly developed yeast estrogen screen. For creation of the new reporter plasmid YRpE2-GFP the whole sequence of YRpE2 except the coding sequence of the lac Z-Gene was amplified by PCR with primers creating a BglII-and a NotI-sticky end at the 5 prime end and the 3 prime end, respectively. The coding sequence for red shifted GFP (rsGFP) was amplified by PCR from the plasmid pQBI63 (Qbiogene, Carlsbad, CA) with the corresponding sticky ends. Both fragments were ligated using T4-DNA-ligase and transformed by electroporation into electrocompetent E.coli for amplification. Plasmids were transformed into hyperpermeable yeast cells (Saccharomyces cerevisiae, strain 188 R1) [22] using a standard lithium-acetate method [37]. Transformed yeast cells were selected by growth on Gold medium without tryptophan and uracil. 2.3. Detection of expressed GFP Expression of the GFP reporter was shown by Western blotting. Over night cultures of yeast were first grown on selective Gold medium without tryptophan and uracil, then diluted into the expression medium YPHSM and again grown over night. Cultures were adjusted to OD600 = 0.5, induced with either 1 mM or 100 AM CuSO4 and 10 nM 17hestradiol were added. After four hours of incubation cells were harvested, washed once and resuspended in the double volume of disintegration buffer (20 mM Tris pH 7.4, 150 mM NaCl, 2 mM DTT, 10% glycerol, 1% Tween 20, 1 ml/20 g protease inhibitor cocktail). Glass beads were added to the yeast cells and disintegration was performed by vortexing three times for 30 s with a rest on ice for 15 s between the intervals. Immediately after disintegration yeast cell extracts were loaded onto a NuPage 4–12% Bis–Tris gel (Invitrogen) using the MES buffer system and SDS-PAGE was performed according to the supplier. Samples were transferred to a Protran nitrocellulose transfer membrane (Schleicher and Schu¨ll, Du¨ren, Germany) by electroblotting. The blot was incubated with an anti-GFP antibody (Santa Cruz Biotechnology, Santa Cruz, CA) as a primary antibody and an anti-mouse IgG alkaline phospatase conjugate (Sigma, St. Louis, MO) as a secondary antibody.
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Ubiquitin
(A)
ampR
ERalpha
YEpE12 7980 bp
TRP1
lacZ
(B)
YRpE2
ampR
11055 bp 5'-CYC1 ERE
URA3
GFP
(C)
5'-CYC1 ERE
YRpE2-GFP 8612 bp ampR URA3
Fig. 1. Maps of the plasmids used in the yeast estrogen screens. (A) expression plasmid YEpE12 coding for ERa, (B) reporter plasmid YRpE2 carrying the lacZ gene coding for h-galactosidase, (C) reporter plasmid YRpE2-GFP carrying the gene for GFP expression.
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2.4. FACS-analysis For fluorescence measurements on a fluorescence activated cell sorter different clones of yeast containing the expression plasmid and the GFP-reporter plasmid were grown on selective medium over night. On the next day the OD600 of the cultures was adjusted to 0.4 and to each clone either zero, 0.5 or 10 nM 17h-estradiol and 10 AM CuSO4 were added. Additionally a yeast clone without addition of 17h-estradiol and without CuSO4-induction and one clone without any plasmids were used as controls. After an induction period of four hours OD600 values of the yeast clones were measured and an equivalent corresponding to OD600 = 1 was taken for analysis. After washing the cells were resuspended in 1 ml PBS and fluorescence was read on a FACSCalibur instrument (Becton Dickinson, New York, USA). 2.5. Transactivation assay The procedure of the yeast transactivation assay using h-galactosidase as a reporter is described in detail in [33]. Shortly, standard substance 17h-estradiol or sample as well as CuSO4 were added to a yeast culture with the OD600 adjusted to 0.4. DMSO alone was used as a blank. A calibration curve with 17h-estradiol was performed within each test run and each determination was performed in duplicates. After four hours of incubation cells were harvested, washed in lacZ-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, pH 7.0, 10 mM KCl, 1 mM MgSO4, 1 mM DTT) and disintegrated with glass beads. Of the clear cell lysates h-galactosidase activity and protein concentration were determined. The specific enzyme activity was expressed in Miller units which take the amount of total protein into account. They are defined as follows: Miller units¼
OD405 1 sample volume protein assay 4 4 41000 Ag of protein=ml Dt sample volume h gal assay
ð1Þ where Dt (given in minutes) is the incubation time of the h-galactosidase assay at 37 8C. 2.6. Transactivation assay using GFP as a reporter gene For each test run a new overnight culture was diluted to OD600 = 0.4 before use. 5 Al of the different standard concentrations as well as 45 Al of DMSO were added to 5 ml of the diluted yeast culture. Expression of ERa was induced by addition of 10 AM CuSO4. DMSO alone was used as a blank. A calibration curve with 17h-estradiol was performed within each test run. Determinations were carried out in duplicates. After 4 h of incubation at 30 8C cells were harvested by centrifugation at 2500 rpm for 5 min and washed in 0.5 ml washing buffer (20 mM Tris pH 7.4, 150 mM NaCl, 10% glycerol). Cells were resuspended in 500 Al washing buffer and 100 Al of each vial were transferred into a black microplate. Fluorescence of yeast cells was measured at an excitation wavelength of 473
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nm and an emission wavelength of 509 nm on a Spectramax Gemini XS fluorimeter (Molecular Devices, Sunnyvale, CA). 2.7. Down-scaling of fluorescent yeast transactivation assay 2.5 Al of standard substance 17h-estradiol or test sample as well as 10 AM CuSO4 were transferred into a black microplate with transparent bottom. Final concentrations of standard substance in the wells were the same as also used for the larger scale of the assay. 100 Al of a diluted overnight yeast culture (OD600 = 0.4) were added into each well and plates were incubated at 30 8C for four, six or 24 h on a shaker. Determinations were performed in duplicates. After incubation fluorescence of the samples was measured at 473 nm excitation wavelength and 509 nm emission wavelength on a Spectramax Gemini XS fluorimeter (Molecular Devices, Sunnyvale, CA). 2.8. Curve fitting Data of the transactivation assay were fitted using a logistic dose–response model. Calculation was performed with Table Curve 2D software (Jandel Scientific). The function is described as: Y ¼aþ
b
ð2Þ
1 þ ðc=xÞd
Miller units
where the parameter a equals the baseline, b is the difference between the plateau of the curve and the baseline (ligand efficiency) and c gives the transition center of the curve (ligand potency), which is the concentration that causes 50% efficiency. d describes the transition zone and is a measure for positive or negative cooperativity [38]. A schematic description of the logistic dose response function is shown in Fig. 2. By using this calibration curve the estrogenic activity of the samples can be expressed in equivalents of the standard substance 17h-estradiol.
b c
17β-Estradiol [mol/l] a
Fig. 2. Schematic drawing of a logistic dose response curve.
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3. Results and discussion A recombinant yeast containing two episomal plasmids was constructed to assess the estrogenic activity of various compounds. The yeast estrogen screen used in our lab usually [8] which employs h-galactosidase as a reporter was changed to utilize green fluorescent protein (GFP) instead. The aim of this study was to establish a test system for the fast and accurate analysis of the estrogenic activity of a large number of samples in relatively short time. After replacement of the lacZ-gene by the gene for red-shifted GFP in the reporter plasmid YRpE2 (see materials and methods and Fig. 1) the expression of the reporter gene product was tested by several methods. In this reporter system the amount of expressed GFP is dependent on the concentration of estrogenically active substance added to the culture and on the addition of CuSO4 which is used as an inductor substance for the expression of ERa. We added two different concentrations of CuSO4 (1 mM and 100 AM) and a constant high concentration of 17h-estradiol (10 nM) to a yeast culture and induced it for four hours. The high 17h-estradiol concentration was chosen to ensure a high GFP expression in the cells that is sufficient for detection by Western blot analysis (Fig. 3). On the Western blot of the yeast cell extracts a difference between the intensities of the two bands cannot be seen. Both inductor concentrations are adequate to induce the expression of ERa and subsequently the reporter gene can be transactivated and expressed in a detectable amount. Simple inspection of induced cells on a confocal laser microscope with or without addition of 17h-estradiol showed that the expressed GFP was functional (Fig. 4). In the presence of 17h-estradiol most of the yeast cells emitted green fluorescence. The differences in fluorescence emission of various yeast clones were assessed by measurement of fluorescence on a fluorimeter and by FACS analysis. Different yeast clones transformed with the YEpE12 expression plasmid and the YRpE2-GFP reporter plasmid were supplemented with either zero, with 0.5 nM or with 10 nM 17h-estradiol.
1
2
3
4
5 [kDa] 98 62 49 38 28 17 14 6
Fig. 3. Western Blot analysis of yeast cell extracts. Cells induced with (1) 1 mM and (2) 100 AM CuSO4. (3) negative control: cell extract of yeast without plasmids. (4) positive control: purified GFP. (5) Marker See Blue Plus 2 (Invitrogen).
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Fig. 4. Fluorescence microscope pictures of yeast cells induced with 10 AM CuSO4. Cells in (A) are without 17hestradiol, in (B) cells are treated with 0.5 nM 17h-estradiol. The left part of the pictures shows all cells, the right part shows those cells that emit fluorescence.
Fluorescence of the cell suspensions was measured after an incubation time of four hours and normalized for cell density. All clones showed rising fluorescence emission with rising 17h-estradiol concentrations. Thus, it could be shown that the amount of expressed GFP is dependent on the 17h-estradiol concentration (Fig. 5A). Different yeast clones showed different levels of GFP expression and a clone with a high induced fluorescence level and a low basal GFP expression was chosen for further experiments. Furthermore a strong shift of population to higher fluorescence emission could be observed by FACS-analysis. The shift is also related to 17h-estradiol concentration which indicates a dose dependent activation of the reporter gene (Fig. 5B). As also shown by other groups [11,39,40], these results indicate that GFP functions as a reporter gene for transactivation assays in yeast. Fluorescence emission of yeast cells is dependent on the concentration of 17h-estradiol. Since the h-gal reporter system has been in use for at least eight years in routine application and has been described in detail in previous studies [8,23,29,30,32– 34,36,41] a thorough evaluation was made to show if the new reporter system could generate data equivalent to the yeast screen currently in use. In a previous paper we have shown that transactivation of a reporter gene can be described by a logistic dose
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(A)
Fluorescence units / OD
3000
0 nM E2 0.5 nM E2 10 nM E2
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0 0
1
2
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9
(B)
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Clone number
Cells w/o CuSO4, w/o E2 Cells with 10 M CuSO4, w/o E2 Cells with 10 M CuSO4, 0.5 nM E2 Cells with 10 M CuSO4, 10 nM E2
0
Events
Black line: Dark gray line: Dotted line: Light gray line:
100
101
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FL2-H (GFP) Fig. 5. (A) Fluorescence emission of different clones of yeast treated with 0 nM, 0.5 nM or 10 nM 17h-estradiol and induced with 10 AM CuSO4. (B) Fluorescence emission of yeast cells induced or not induced with 10 AM CuSO4 and treated with different amounts of 17h-estradiol. With higher 17h-estradiol concentration the population is shifted to higher fluorescence emission.
response function [38], see Eq. (2). This function contains four parameters which describe the dose response behavior. The meaning of each parameter has been briefly described in the materials and methods section. In Fig. 6 the four parameters characterizing the logistic dose functions obtained with h-galactosidase as a reporter over a time range of approximately three years are plotted versus the number of experiments. These plots show accuracy and reproducibility. The yeast estrogen screen employing h-galactosidase as a reporter gene is very sensitive. The detection limit of the assay was defined as the mean value of the blank plus the three fold standard deviation of the blank. As a blank the pure
V. Beck et al. / J. Biochem. Biophys. Methods 64 (2005) 19–37
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(A)
Miller units
100000
10000
Parameter a Parameter b
1000
100 0
20
40
60
80
100
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Number of experiment
(B) 1,8e-9
0 -1
1,4e-9 -2
1,2e-9 1,0e-9
-3
8,0e-10
-4
6,0e-10
Parameter d
Parameter c [M]
1,6e-9
Parameter c Parameter d
-5
4,0e-10 -6
2,0e-10 0,0
-7 0
20
40
60
80
100
120
Number of experiment Fig. 6. Parameters of the logistic dose response curves obtained over a time frame of approximately three years in 127 experiments. (A) Parameter a is the baseline, b is the plateau of the curve, (B) c is the potency and d is the transition width.
solvent (DMSO) was taken and for 17h-estradiol a detection limit of 0.1 nM could be determined [8]. The sensitivity of the newly developed transactivation assay determined by analysis of various concentrations of 17h-estradiol was in the same range as for the conventional yeast estrogen screen (Figs. 7 and 8). The same low amounts of the standard substance can be detected. The potency of 17h-estradiol, which is the concentration necessary to reach half maximal transactivation, is also in the same range for both test systems. The performance of the estrogen screen using GFP as a reporter was compared with the performance of the established test system used in our lab for routine analysis. The aim of the study was to show that the newly developed yeast screen worked at least as well as the established routine estrogen screen. Various concentrations of fourteen different compounds belonging to different chemical classes (17h-estradiol, estrone,
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(A)
Relative Response [%]
250
200
17β-Estradiol Estrone Estriol 17α-Ethinylestradiol 17α-Estradiol Diethylstilbestrol Mestranol
150
100
50
0 1e-14 1e-13 1e-12 1e-11 1e-10 1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
Concentration [M]
(B) 250
Relative Response [%]
200
17β-Estradiol Coumestrol Daidzein Formononetin Genistein Biochanin A 4-Nonylphenol Zearalenone
150
100
50
0 1e-11 1e-10
1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
1e-3
Concentration [M] Fig. 7. Logistic dose response curves for various estrogenically active substances obtained in the yeast estrogen screen with h-galactosidase as a reporter. The response was related to E2. (A) shows the curves of various natural and synthetic estrogens (B) shows various phytoestrogens, the xenoestrogen 4-nonylphenol and the fungal estrogen zearalenone.
estriol, 17a-estradiol, 17a-ethinylestradiol, mestranol, diethylstilbestrol, zearalenone, 4nonylphenol, coumestrol, genistein, daidzein, formononetin and biochanin A) were analyzed with both test systems and logistic dose response curves were generated (Figs. 7 and 8). Potencies of the substances obtained in the tests were very similar using either the conventional yeast assay with h-galactosidase or the newly developed assay with GFP. Most of the potencies were also in accordance to values found in previous studies
V. Beck et al. / J. Biochem. Biophys. Methods 64 (2005) 19–37
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(A) 140
Relative Response [%]
120
17β-Estradiol Estrone Estriol Mestranol 17α-Estradiol 17α-Ethinylestradiol Diethylstilbestrol
100 80 60 40 20 0 1e-14 1e-13 1e-12 1e-11 1e-10 1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
Concentration [M]
(B) 140
Relative Response [%]
120
17β-Estradiol Biochanin A Daidzein Formononetin Genistein Coumestrol 4-Nonylphenol Zearalenone
100 80 60 40 20 0 1e-11 1e-10
1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
1e-3
Concentration [M] Fig. 8. Logistic dose response curves for various estrogenically active substances obtained in the yeast estrogen screen with GFP as a reporter. The response was related to E2. (A) shows the curves of various natural and synthetic estrogens (B) shows various phytoestrogens, the xenoestrogen 4-nonylphenol and the fungal estrogen zearalenone.
[8]. To show the good agreement of the values obtained in the different assay systems the potency of the h-gal assay was plotted against the potency of the GFP-assay (Fig. 9). Most of the values are within the dashed lines y = 3x and 3y = x which surround the straight line y = x. This means that the potencies in both test systems are more or less identical. Values outside the dashed lines are more than three times higher or lower in one assay system than in the other. With these results it could be shown that the
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17β-Estradiol Estrone Estriol 4-Nonylphenol Coumestrol Biochanin A Daidzein Genistein Formononetin Mestranol Diethylstilbestrol Zearalenon 17α-Estradiol 17α-Ethinylestradiol
1e-5
Potency GFP-assay
1e-6
1e-7
1e-8
1e-9
1e-10
1e-11 1e-11
1e-10
1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
Potency β-Gal-assay Fig. 9. Comparison of potencies measured with test systems using h-gal or GFP as reporter. Potencies obtained in the h-gal yeast assay are plotted on the abscissa and potencies obtained in the GFP yeast assay were plotted on the ordinate. The dashed lines x = 3y and 3x = y are also shown. Symbols between the lines designate similar estrogenic activity in both test systems. Values outside the lines show potencies that are at least three times higher or lower in one assay than in the other.
applicable concentration range and the sensitivity are the same for both test systems and that the potencies of various substances are reproducible in the yeast transactivation assay, independent of the reporter plasmid used. In order to make the yeast estrogen screen more suitable for large sample sizes and fast analysis of estrogenic activities the test was down-scaled to be performed in microplates. The same concentrations of inductor substance and of 17h-estradiol standards were used as established for the larger scale of the test. Fluorescence measurements were performed on a microplate fluorescence reader after incubation for four, six and 24 h without any further sample treatment. Various incubation times for the yeast estrogen screen in microplates were tested. It was found that fluorescence measurements after six hours of incubation led to results that could be better fitted to a logistic dose response curve than those obtained after an incubation time of only four hours. Longer incubation times up to 24 h did not improve the quality of the resulting logistic dose response curve fits. Logistic dose response curves obtained in microplates are shown in Fig. 10. The incubation time of six hours did not alter the overall time needed for the assay compared to the h-gal yeast estrogen screen because the timeconsuming steps of cell disintegration, determination of h-gal activity and protein concentration after an incubation time of four hours could be omitted when GFP was used as a reporter gene. A comparison of potencies determined by the microplate assay and the
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(A) 120
Relative Response [%]
100
17β-Estradiol Estrone Estriol Mestranol 17α-Ethinylestradiol 17α-Estradiol Diethylstilbestrol
80
60
40
20
0 1e-15 1e-141e-13 1e-121e-11 1e-10 1e-9 1e-8 1e-7 1e-6 1e-5 1e-4
Concentration [M]
(B) 120
Relative Response [%]
100
17β-Estradiol Biochanin A Daidzein Formononetin Genistein Coumestrol 4-Nonylphenol Zearalenone
80
60
40
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0 1e-11
1e-10
1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
1e-3
Concentration [M] Fig. 10. Logistic dose response curves for various estrogenically active substances obtained in microplates. (A) shows the curves of various natural and synthetic estrogens, (B) shows various phytoestrogens, the xenoestrogen 4-nonylphenol and the fungal estrogen zearalenone.
conventional protocol in tubes is shown in Fig. 11. Potencies of the logistic dose response curves of various compounds were determined and most of the values were in the same range as also found in the larger scale of the assay. However, the potencies of the estrogens 17a-estradiol, 17a-ethinylestradiol and mestranol obtained in the microplate assay were about two orders of magnitude higher than those obtained in the larger scale of the assay.
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17β-Estradiol Estrone Estriol 4-Nonylphenol Coumestrol Biochanin A Daidzein Genistein Formononetin Mestranol Diethylstilbestrol Zearalenon 17α-Estradiol 17α-Ethinylestradiol
1e-5
Potency GFP-assay
1e-6
1e-7
1e-8
1e-9
1e-10
1e-11 1e-11
1e-10
1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
Potency micro-YES Fig. 11. Comparison of potencies measured with micro plate system and large scale system using GFP as reporter. Potencies obtained in microplate assay are plotted on the abscissa and potencies obtained large scale assay were plotted on the ordinate. The dashed lines x = 3y and 3x = y are also shown. Symbols between the lines designate similar estrogenic activity in both test systems. Values outside the lines show potencies that are at least three times higher or lower in one assay than in the other.
This indicates that lower concentrations of these substances can be detected in the microplate scale. Most of the potencies determined in the yeast assay in microplate scale are in accordance to the values obtained by other investigators using a similar test system [39]. The potency for the phytoestrogen coumestrol obtained in our newly developed assay was about one order of magnitude higher and the potencies for 17a-estradiol, 17aethinylestradiol and mestranol were about two orders of magnitude lower than those determined by Bovee et al. [39]. It is evident that the test system is more sensitive for these substances. We could also determine a potency value for the isoflavone daidzein, whereas Bovee et al. [39] did not find any response of ERa-expressing yeast to this compound. The high reproducibility of the microplate assay could be shown by performing the yeast estrogen screen with the same samples on different days. The parameters of the logistic dose response curves of the 17h-estradiol standard after six hours of incubation resulted in similar values on each day (Fig. 12). All parameters were in a quite narrow range in all assays performed. Parameter b shows the greatest variation of all parameters which might be due to the different age of the yeast culture used for inoculation of the over night culture used for the test. Although cells are freshly grown over night before each test, there are small variations in the number of fluorescing cells. The parameter a which equals the baseline of the curve, however, is not higher. This means that the correlation between the baseline and the plateau of the curve is higher when fresh yeast is used for inoculation
V. Beck et al. / J. Biochem. Biophys. Methods 64 (2005) 19–37
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100
26 24
Parameter b [fluorescence units]
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(A) 90
22 80
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12 10
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4
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6,0e-10
Parameter c Parameter d
3,0e-10 -3 2,0e-10 0
2
4
6
8
10
12
14
16
18
20
Number of experiment Fig. 12. Parameters of the logistic dose response curves of different batches after an incubation time of six hours obtained in a GFP yeast screen in microplates. (A) shows parameter a (baseline) and b (plateau of the curve), (B) shows parameter c (potency) and d (transition width).
of the over night culture and thus the range within which estrogenic activity can be detected is higher. Parameters c and d are quite constant over the time. Small variations are due to the biological nature of the test. The absolute range between baseline and plateau of the logistic dose response curve is higher in the yeast assay using h-gal as a reporter than in the GFP-microplate-assay. Nevertheless, good fits to the logistic dose response function can be obtained in the small scale assay.
4. Conclusion It could be shown that GFP functions as a reporter gene for the yeast estrogen screen and that the use of GFP as a reporter is a useful tool to make the assay procedure faster and
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easier. As there are less pipeting steps than in the conventional yeast estrogen screen with h-gal as a reporter gene, there are also less sources of error. In a routine analysis procedure the newly developed GFP-yeast assay can be used when high sample throughput in short time is required. Many microplates can be incubated and measured almost simultaneously and on each plate the equivalent estrogenic activity of at least five samples can be analyzed. As the yeast suspension volume per well is only 100 Al compared to 5 ml in the h-gal yeast assay, the GFP-assay is much cheaper regarding media requirements. As the potencies of all of the tested substances were in accordance to the conventional yeast screen, we concluded that the newly developed transactivation assay performed well enough to substitute the conventional h-gal transactivation assay.
References [1] Fang H, Tong W, Perkins R, Soto AM, Prechtl NV, Sheehan DM. Quantitative comparisons of in vitro assays for estrogenic activities. Environ Health Perspect 2000;108:723 – 9. [2] Gutendorf B, Westendorf J. Comparison of an array of in vitro assays for the assessment of the estrogenic potential of natural and synthetic estrogens, phytoestrogens and xenoestrogens. Toxicology 2001;166:79 – 89. [3] Matthews J, Celius T, Halgren R, Zacharewski T. Differential estrogen receptor binding of estrogenic substances: a species comparison. J Steroid Biochem Mol Biol 2000;74:223 – 34. [4] Morito K, Hirose T, Kinjo J, Hirakawa T, Okawa M, Nohara T, et al. Interaction of phytoestrogens with estrogen receptors alpha and beta. Biol Pharm Bull 2001;24:351 – 6. [5] Soto AM, Sonnenschein C, Chung KL, Fernandez MF, Olea N, Serrano FO. The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ Health Perspect 1995;103:113 – 22. [6] Gaido KW, Leonard LS, Lovell S, Gould JC, Babai D, Portier CJ, et al. Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. Toxicol Appl Pharmacol 1997;143:205 – 12. [7] Byford JR, Shaw LE, Drew MGB, Pope GS, Sauer MJ, Darbre PD. Oestrogenic activity of parabens in MCF7 human breast cancer cells. J Steroid Biochem Mol Biol 2002;80:49 – 60. [8] Jungbauer A, Beck V. Yeast reporter system for rapid determination of estrogenic activity. J Chromatogr B Analyt Technol Biomed Life Sci 2002;777:167. [9] Safe S, Connor K, Gaido K. Methods for xenoestrogen testing. Toxicol Lett 1998;102–103:665 – 70. [10] Sugawara T, Nakajima A, Nomura E. Development of a simple screening system for endocrine disruptors. Med Sci Monit 2002;8:BR431 – 8. [11] Bovee TFH, Helsdingen RJR, Koks PD, Kuiper HA, Hoogenboom RLAP, Keijer J. Development of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein. Gene 2004;325:187 – 200. [12] Kojima H, Katsura E, Takeuchi S, Niiyama K, Kobayashi K. Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. Environ Health Perspect 2004;112:524 – 31. [13] Routledge EJ, Sumpter JP. Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environ Toxicol Chem 1996;15:241 – 8. [14] Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-A. Cloning of a novel estrogen receptor expressed in rat prostate and ovary. PNAS 1996;93:5925 – 30. [15] Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, et al. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 1998;139:4252 – 63. [16] Morito K, Aomori T, Hirose T, Kinjo J, Hasegawa J, Ogawa S, et al. Interaction of phytoestrogens with estrogen receptors alpha and beta (II). Biol Pharm Bull 2002;25:48 – 52. [17] Gustafsson JA. Estrogen receptor beta—a new dimension in estrogen mechanism of action. J Endocrinol 1999;163:379 – 83.
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[18] Nilsson S, Gustafsson JA. Estrogen receptor transcription and transactivation: basic aspects of estrogen action. Breast Cancer Res 2000;2:360 – 6 Epub 2000 Jul 13. [19] Graumann K, Jungbauer A. Quantitative assessment of complex formation of nuclear-receptor accessory proteins. Biochem J 2000;345:627 – 36. [20] Miller S, Kennedy D, Thomson J, Han F, Smith R, Ing N, et al. A rapid and sensitive reporter gene that uses green fluorescent protein expression to detect chemicals with estrogenic activity. Toxicol Sci 2000;55:69 – 77. [21] Metzger D, White JH, Chambon P. The human oestrogen receptor functions in yeast. Nature 1988;334:31 – 6. [22] Lyttle CR, Damian-Matsumura P, Juul H, Butt TR. Human estrogen receptor regulation in a yeast model system and studies on receptor agonists and antagonists. J Steroid Biochem Mol Biol 1992;42:677 – 85. [23] Breithofer A, Graumann K, Scicchitano MS, Karathanasis SK, Butt TR, Jungbauer A. Regulation of human estrogen receptor by phytoestrogens in yeast and human cells. J Steroid Biochem Mol Biol 1998;67:421 – 9. [24] Serebriiskii I, Golemis EA. Uses of lacZ to study gene function: evaluation of [beta]-galactosidase assays employed in the yeast two-hybrid system. Anal Biochem 2000;285:1 – 15. [25] March JC, Rao G, Bentley WE. Biotechnological applications of green fluorescent protein. Appl Microbiol Biotechnol 2003;62:303 – 15. [26] Tsien RY. The green fluorescent protein. Annu Rev Biochem 1998;67:509 – 44. [27] Heim R, Prasher D, Tsien R. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. PNAS 1994;91:12501 – 4. [28] Zimmer M. Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev 2002;102:759 – 81. [29] Graumann K, Wittliff JL, Raffelsberger W, Miles L, Jungbauer A, Butt TR. Structural and functional analysis of N-terminal point mutants of the human estrogen receptor. J Steroid Biochem Mol Biol 1996;57:293 – 300. [30] Graumann K, Jungbauer A. Agonistic and synergistic activity of tamoxifen in a yeast model system. Biochem Pharmacol 2000;59:177 – 85. [31] Varshavsky A. The N-end rule. Cell 1992;69:725 – 35. [32] Jisa E, Dornstauder E, Ogawa S, Inoue S, Muramatsu M, Jungbauer A. Transcriptional activities of estrogen receptor alpha and beta in yeast properties of raloxifene. Biochem Pharmacol 2001;62:953 – 61. [33] Beck V, Unterrieder E, Krenn L, Kubelka W, Jungbauer A. Comparison of hormonal activity (estrogen, androgen and progestin) of standardized plant extracts for large scale use in hormone replacement therapy. J Steroid Biochem Mol Biol 2003;84:259 – 68. [34] Dornstauder E, Jisa E, Unterrieder I, Krenn L, Kubelka W, Jungbauer A. Estrogenic activity of two standardized red clover extracts (Menoflavon) intended for large scale use in hormone replacement therapy. J Steroid Biochem Mol Biol 2001;78:67 – 75. [35] Klinge CM, Risinger KE, Watts MB, Beck V, Eder R, Jungbauer A. Estrogenic activity in white and red wine extracts. J Agric Food Chem 2003;51:1850 – 7. [36] Promberger A, Dornstauder E, Fruhwirth C, Schmid ER, Jungbauer A. Determination of estrogenic activity in beer by biological and chemical means. J Agric Food Chem 2001;49:633 – 40. [37] Schiestl RH, Gietz RD. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 1989;16:339 – 46. [38] Jungbauer A, Graumann K. The logistic dose–response function: a robust fitting function for transition phenomena in life sciences. J Clin Ligand Assay 2001;24:270 – 4. [39] Bovee TFH, Helsdingen RJR, Rietjens IMCM, Keijer J, Hoogenboom RLAP. Rapid yeast estrogen bioassays stably expressing human estrogen receptors [alpha] and [beta], and green fluorescent protein: a comparison of different compounds with both receptor types. J Steroid Biochem Mol Biol 2004;91:99 – 109. [40] Cormack RS, Hahlbrock K, Somssich IE. Isolation of putative plant transcriptional coactivators using a modified two-hybrid system incorporating a GFP reporter gene. Plant J 1998;14:685 – 92. [41] Graumann K, Breithofer A, Jungbauer A. Monitoring of estrogen mimics by a recombinant yeast assay: synergy between natural and synthetic compounds? Sci Total Environ 1999;225:69 – 79.
GFP-reporter for a high throughput assay to monitor estrogenic compounds Verena Beck, Angelika Pfitscher, Alois Jungbauer* Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria Received 10 February 2005; received in revised form 21 April 2005; accepted 9 May 2005
Abstract In vitro test systems using yeast cells are a useful tool for the determination of the estrogenic activity of estrogens, phyto- and xeno-estrogens and can be used for monitoring large sample numbers in a routine analysis procedure. Our conventional transactivation assay functions with an expression plasmid expressing estrogen receptor a (ERa) under the control of a copper-inducible CUP1 promoter and a reporter plasmid expressing h-galactosidase under the control of the vitellogenin estrogen response element (ERE). In the novel yeast screen system the lacZ gene in the reporter plasmid was substituted by a gene for green fluorescent protein (GFP). Incubation of yeast with various concentrations of estrogenically active substances led to expression of the reporter gene product GFP in a dose dependent manner. The yeast transactivation assay was further down-scaled to be performed in a microplate scale, which is an important step to facilitate handling of large sample numbers. The sensitivity and reproducibility of the novel test system could be confirmed by analysis of the potencies of various estrogenically active substances. Thus, the newly developed yeast estrogen screen using GFP as a reporter can substitute the assay that has been used for a period of several years. D 2005 Elsevier B.V. All rights reserved. Keywords: 17h-estradiol; Ethinylestradiol; Mestranol; Green fluorescent protein (GFP); Reporter gene; Estrogen receptor a
* Corresponding author. Tel.: +43 1 360066226; fax: +43 1 3697615. E-mail address: [email protected] (A. Jungbauer). 0165-022X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2005.05.001
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1. Introduction The development of rapid and simple in vitro test systems for high-throughput screening for the detection and characterization of substances with estrogenic activity is of interest for the assessment of (a) the potency of newly synthesized drug candidates for treatment of diseases related to estrogens, (b) the potency of plant extracts intended for use as herbal remedies, (c) the potency of metabolites of steroids or polyphenols in urine, feces or plasma and (d) for the analysis of environmental samples and xenoestrogens to perform risk assessment studies. The advantages of in vitro tests are their cost-effectiveness, their high sensitivity and specificity and the high throughput compared to in vivo test systems, although these in vitro systems can never replace in vivo studies. Various in vitro test systems such as receptor binding tests [1–4], cell proliferation assays [1,2,5] or reporter gene assays [6–10] are in use. The latter test system employs the transactivating potential of estrogenically active substances on the hormone receptor [11–13]. Many chemicals exerting estrogenic activity act through the estrogen receptor pathway. The mechanism of action on both known estrogen receptors, estrogen receptor alpha (ERa) and estrogen receptor beta (ERh) [14], is the same, but a substance can have a different transactivational potential on both ERs [4,15,16] which are expressed in different tissues [17,18]. Estrogen receptors belong to the steroid–thyroid hormone receptor superfamily and are located in the nucleus of the cell. In their inactive form they are associated to heat shock proteins in a multiprotein complex [19]. Binding of substances to the ligand binding pocket of the estrogen receptor leads to its activation. The activated, ligandbound estrogen receptor dissociates from the multiprotein complex, dimerizes and moves to the nucleus, where it can bind to an estrogen response element (ERE). The EREs are specific DNA sequences in the promoter region of estrogen target genes. The transcription of these genes is mediated by binding of the activated estrogen receptor dimer to their promoter region. Thus, estrogen receptors act as ligand activated transcription factors. Chemical analyses of estrogenically active substances or binding experiments to the receptor are not always suitable to assess the biological effect of those compounds. Thus, many in vitro reporter gene test systems have been developed that imitate the mechanism of estrogen action via the estrogen receptor pathway and that are suitable to assess the estrogenic potential of a variety of substances. These test systems are either performed in mammalian cell lines expressing the estrogen receptor such as the breast cancer cell line MCF-7 [2,20] or in recombinant yeast strains such as Saccharomyces cerevisiae. Metzger et al. [21] have shown that estrogen receptor functions in yeast. This is the basic finding on which all other yeast reporter systems are based. The test systems developed subsequently either vary in expression plasmid, hormone response element, reporter gene and yeast strain [6,10,11,13,22]. The main advantages of the yeast test systems compared to mammalian cell systems are the cheaper media components and faster growth of yeast, as well as the robustness towards toxic effects such as endotoxins or solvents. With rising sample numbers also the shorter time that is needed for performing the assay is an important factor.
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Many investigators have developed transactivation assays in yeast for their requirements and most of them employ the lacZ gene, which is the gene coding for the enzyme h-galactosidase, as an estrogen-responsive reporter gene [13,22,23]. Concentration of expressed h-galactosidase is correlated to the transactivation potential of the tested compound. Activity of h-galactosidase is detected by photometric measurement of a colored reaction product that develops after cleavage of a colorless substrate [24]. However, for determination of h-galactosidase activity yeast cells have to be disrupted which is a time-consuming procedure. Furthermore cell disruption demands some additional handling steps in the analysis procedure which is a major source of error. An effective possibility to make the reporter gene assay procedure faster is the implementation of green fluorescent protein (GFP) instead of h-galactosidase as a reporter gene. GFP is a protein used very often for various biotechnological studies due to its many application possibilities [25]. The GFP protein is derived from the jelly fish Aequorea victoria and emits green fluorescing light which can be measured directly without cell disintegration [26]. The chromophore of GFP is formed by intramolecular cyclization and subsequent dehydrogenation without adding any cofactors [27]. The green fluorescent protein itself is rather small, highly soluble and stable in a broad pH range [28]. In our lab a yeast transactivation assay for the determination of the estrogenic activity of various samples has been used [8,22,29,30]. The system is based on ubiquitin estrogen receptor fusions yielding into a stably expressed hormone receptor [29]. Subsequent modification of the N-terminus according to the N-end rule from Varshavsky [31] showed effects of receptor expression level and activation function-1 (AF-1) domain on transactivation. This test system has been in use for many years and has been applied for the analysis of various kinds of sample matrices such as the selective estrogen receptor modulators (SERMs) raloxifene [32] and tamoxifen [30], phytoestrogenic preparations [33,34], food samples [35,36] and water samples. In order to simplify the assay procedure the reporter gene lacZ has been replaced by GFP and the performance of both test systems regarding sensitivity and reproducibility was compared. To make the test suited for the analysis of an even larger sample number with smaller volumes it was down-scaled to a microplate scale and it could be shown that results were in agreement with the results obtained in a larger scale.
2. Materials and methods 2.1. Chemicals Buffer reagents, dimethylsulfoxide (DMSO) and o-nitrophenyl-h-galactopyranoside (ONPG) were purchased from Merck (Darmstadt, Germany) or Sigma Aldrich (St. Louis, MO). For yeast media preparation, yeast nitrogen base was obtained from Difco (Franklin Lakes, NJ), amino acids from Serva Feinbiochemica (Heidelberg, Germany) and from Sigma Aldrich (St. Louis, MO). Enzymes for molecular biology were purchased from Fermentas GmbH (St. Leon-Rot, Germany) and electrocompetent E. coli were obtained from Invitrogen (Lofer, Austria). Protease inhibitor cocktail for fungal and yeast cells was
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purchased from Sigma Aldrich (St. Louis, MO). 17h-estradiol, estrone, estriol, 17aestradiol, 17a-ethinylestradiol, diethylstilbestrol, coumestrol, zearalenone and isoflavones were obtained from Sigma Aldrich (St. Louis, MO), 4-nonylphenol (4-NP) technical mixture (PestanalR) was obtained from Riedel-de-Haen, mestranol was purchased from ICN Biomedicals (Eschwege, Germany). Glass beads were purchased from Merck (Darmstadt, Germany). The solvents used were HPLC grade. Black microplates were obtained from Corning (Schiphol-Rijk, The Netherlands), microplates with black walls and transparent bottom were purchased from Perkin Elmer (Boston, MA). 2.2. Construction of plasmids The expression plasmid YEpE12 carrying the human estrogen receptor alpha (ERa), the CUP promoter and a tryptophan–marker and the reporter plasmid YRpE2 (Fig. 1) with two copies of the vitellogenin ERE, the iso-1-cytochrome c (CYC1) promoter in fusion to the lacZ and an uracil auxotrophy marker were a gift from C. R. Lyttle [22]. The expression plasmid YEpE12 was used in the conventional and in the newly developed yeast estrogen screen. For creation of the new reporter plasmid YRpE2-GFP the whole sequence of YRpE2 except the coding sequence of the lac Z-Gene was amplified by PCR with primers creating a BglII-and a NotI-sticky end at the 5 prime end and the 3 prime end, respectively. The coding sequence for red shifted GFP (rsGFP) was amplified by PCR from the plasmid pQBI63 (Qbiogene, Carlsbad, CA) with the corresponding sticky ends. Both fragments were ligated using T4-DNA-ligase and transformed by electroporation into electrocompetent E.coli for amplification. Plasmids were transformed into hyperpermeable yeast cells (Saccharomyces cerevisiae, strain 188 R1) [22] using a standard lithium-acetate method [37]. Transformed yeast cells were selected by growth on Gold medium without tryptophan and uracil. 2.3. Detection of expressed GFP Expression of the GFP reporter was shown by Western blotting. Over night cultures of yeast were first grown on selective Gold medium without tryptophan and uracil, then diluted into the expression medium YPHSM and again grown over night. Cultures were adjusted to OD600 = 0.5, induced with either 1 mM or 100 AM CuSO4 and 10 nM 17hestradiol were added. After four hours of incubation cells were harvested, washed once and resuspended in the double volume of disintegration buffer (20 mM Tris pH 7.4, 150 mM NaCl, 2 mM DTT, 10% glycerol, 1% Tween 20, 1 ml/20 g protease inhibitor cocktail). Glass beads were added to the yeast cells and disintegration was performed by vortexing three times for 30 s with a rest on ice for 15 s between the intervals. Immediately after disintegration yeast cell extracts were loaded onto a NuPage 4–12% Bis–Tris gel (Invitrogen) using the MES buffer system and SDS-PAGE was performed according to the supplier. Samples were transferred to a Protran nitrocellulose transfer membrane (Schleicher and Schu¨ll, Du¨ren, Germany) by electroblotting. The blot was incubated with an anti-GFP antibody (Santa Cruz Biotechnology, Santa Cruz, CA) as a primary antibody and an anti-mouse IgG alkaline phospatase conjugate (Sigma, St. Louis, MO) as a secondary antibody.
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Ubiquitin
(A)
ampR
ERalpha
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YRpE2
ampR
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YRpE2-GFP 8612 bp ampR URA3
Fig. 1. Maps of the plasmids used in the yeast estrogen screens. (A) expression plasmid YEpE12 coding for ERa, (B) reporter plasmid YRpE2 carrying the lacZ gene coding for h-galactosidase, (C) reporter plasmid YRpE2-GFP carrying the gene for GFP expression.
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2.4. FACS-analysis For fluorescence measurements on a fluorescence activated cell sorter different clones of yeast containing the expression plasmid and the GFP-reporter plasmid were grown on selective medium over night. On the next day the OD600 of the cultures was adjusted to 0.4 and to each clone either zero, 0.5 or 10 nM 17h-estradiol and 10 AM CuSO4 were added. Additionally a yeast clone without addition of 17h-estradiol and without CuSO4-induction and one clone without any plasmids were used as controls. After an induction period of four hours OD600 values of the yeast clones were measured and an equivalent corresponding to OD600 = 1 was taken for analysis. After washing the cells were resuspended in 1 ml PBS and fluorescence was read on a FACSCalibur instrument (Becton Dickinson, New York, USA). 2.5. Transactivation assay The procedure of the yeast transactivation assay using h-galactosidase as a reporter is described in detail in [33]. Shortly, standard substance 17h-estradiol or sample as well as CuSO4 were added to a yeast culture with the OD600 adjusted to 0.4. DMSO alone was used as a blank. A calibration curve with 17h-estradiol was performed within each test run and each determination was performed in duplicates. After four hours of incubation cells were harvested, washed in lacZ-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, pH 7.0, 10 mM KCl, 1 mM MgSO4, 1 mM DTT) and disintegrated with glass beads. Of the clear cell lysates h-galactosidase activity and protein concentration were determined. The specific enzyme activity was expressed in Miller units which take the amount of total protein into account. They are defined as follows: Miller units¼
OD405 1 sample volume protein assay 4 4 41000 Ag of protein=ml Dt sample volume h gal assay
ð1Þ where Dt (given in minutes) is the incubation time of the h-galactosidase assay at 37 8C. 2.6. Transactivation assay using GFP as a reporter gene For each test run a new overnight culture was diluted to OD600 = 0.4 before use. 5 Al of the different standard concentrations as well as 45 Al of DMSO were added to 5 ml of the diluted yeast culture. Expression of ERa was induced by addition of 10 AM CuSO4. DMSO alone was used as a blank. A calibration curve with 17h-estradiol was performed within each test run. Determinations were carried out in duplicates. After 4 h of incubation at 30 8C cells were harvested by centrifugation at 2500 rpm for 5 min and washed in 0.5 ml washing buffer (20 mM Tris pH 7.4, 150 mM NaCl, 10% glycerol). Cells were resuspended in 500 Al washing buffer and 100 Al of each vial were transferred into a black microplate. Fluorescence of yeast cells was measured at an excitation wavelength of 473
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nm and an emission wavelength of 509 nm on a Spectramax Gemini XS fluorimeter (Molecular Devices, Sunnyvale, CA). 2.7. Down-scaling of fluorescent yeast transactivation assay 2.5 Al of standard substance 17h-estradiol or test sample as well as 10 AM CuSO4 were transferred into a black microplate with transparent bottom. Final concentrations of standard substance in the wells were the same as also used for the larger scale of the assay. 100 Al of a diluted overnight yeast culture (OD600 = 0.4) were added into each well and plates were incubated at 30 8C for four, six or 24 h on a shaker. Determinations were performed in duplicates. After incubation fluorescence of the samples was measured at 473 nm excitation wavelength and 509 nm emission wavelength on a Spectramax Gemini XS fluorimeter (Molecular Devices, Sunnyvale, CA). 2.8. Curve fitting Data of the transactivation assay were fitted using a logistic dose–response model. Calculation was performed with Table Curve 2D software (Jandel Scientific). The function is described as: Y ¼aþ
b
ð2Þ
1 þ ðc=xÞd
Miller units
where the parameter a equals the baseline, b is the difference between the plateau of the curve and the baseline (ligand efficiency) and c gives the transition center of the curve (ligand potency), which is the concentration that causes 50% efficiency. d describes the transition zone and is a measure for positive or negative cooperativity [38]. A schematic description of the logistic dose response function is shown in Fig. 2. By using this calibration curve the estrogenic activity of the samples can be expressed in equivalents of the standard substance 17h-estradiol.
b c
17β-Estradiol [mol/l] a
Fig. 2. Schematic drawing of a logistic dose response curve.
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3. Results and discussion A recombinant yeast containing two episomal plasmids was constructed to assess the estrogenic activity of various compounds. The yeast estrogen screen used in our lab usually [8] which employs h-galactosidase as a reporter was changed to utilize green fluorescent protein (GFP) instead. The aim of this study was to establish a test system for the fast and accurate analysis of the estrogenic activity of a large number of samples in relatively short time. After replacement of the lacZ-gene by the gene for red-shifted GFP in the reporter plasmid YRpE2 (see materials and methods and Fig. 1) the expression of the reporter gene product was tested by several methods. In this reporter system the amount of expressed GFP is dependent on the concentration of estrogenically active substance added to the culture and on the addition of CuSO4 which is used as an inductor substance for the expression of ERa. We added two different concentrations of CuSO4 (1 mM and 100 AM) and a constant high concentration of 17h-estradiol (10 nM) to a yeast culture and induced it for four hours. The high 17h-estradiol concentration was chosen to ensure a high GFP expression in the cells that is sufficient for detection by Western blot analysis (Fig. 3). On the Western blot of the yeast cell extracts a difference between the intensities of the two bands cannot be seen. Both inductor concentrations are adequate to induce the expression of ERa and subsequently the reporter gene can be transactivated and expressed in a detectable amount. Simple inspection of induced cells on a confocal laser microscope with or without addition of 17h-estradiol showed that the expressed GFP was functional (Fig. 4). In the presence of 17h-estradiol most of the yeast cells emitted green fluorescence. The differences in fluorescence emission of various yeast clones were assessed by measurement of fluorescence on a fluorimeter and by FACS analysis. Different yeast clones transformed with the YEpE12 expression plasmid and the YRpE2-GFP reporter plasmid were supplemented with either zero, with 0.5 nM or with 10 nM 17h-estradiol.
1
2
3
4
5 [kDa] 98 62 49 38 28 17 14 6
Fig. 3. Western Blot analysis of yeast cell extracts. Cells induced with (1) 1 mM and (2) 100 AM CuSO4. (3) negative control: cell extract of yeast without plasmids. (4) positive control: purified GFP. (5) Marker See Blue Plus 2 (Invitrogen).
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Fig. 4. Fluorescence microscope pictures of yeast cells induced with 10 AM CuSO4. Cells in (A) are without 17hestradiol, in (B) cells are treated with 0.5 nM 17h-estradiol. The left part of the pictures shows all cells, the right part shows those cells that emit fluorescence.
Fluorescence of the cell suspensions was measured after an incubation time of four hours and normalized for cell density. All clones showed rising fluorescence emission with rising 17h-estradiol concentrations. Thus, it could be shown that the amount of expressed GFP is dependent on the 17h-estradiol concentration (Fig. 5A). Different yeast clones showed different levels of GFP expression and a clone with a high induced fluorescence level and a low basal GFP expression was chosen for further experiments. Furthermore a strong shift of population to higher fluorescence emission could be observed by FACS-analysis. The shift is also related to 17h-estradiol concentration which indicates a dose dependent activation of the reporter gene (Fig. 5B). As also shown by other groups [11,39,40], these results indicate that GFP functions as a reporter gene for transactivation assays in yeast. Fluorescence emission of yeast cells is dependent on the concentration of 17h-estradiol. Since the h-gal reporter system has been in use for at least eight years in routine application and has been described in detail in previous studies [8,23,29,30,32– 34,36,41] a thorough evaluation was made to show if the new reporter system could generate data equivalent to the yeast screen currently in use. In a previous paper we have shown that transactivation of a reporter gene can be described by a logistic dose
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(A)
Fluorescence units / OD
3000
0 nM E2 0.5 nM E2 10 nM E2
2000
1000
0 0
1
2
3
4
5
6
7
8
9
(B)
100
Clone number
Cells w/o CuSO4, w/o E2 Cells with 10 M CuSO4, w/o E2 Cells with 10 M CuSO4, 0.5 nM E2 Cells with 10 M CuSO4, 10 nM E2
0
Events
Black line: Dark gray line: Dotted line: Light gray line:
100
101
102
103
104
FL2-H (GFP) Fig. 5. (A) Fluorescence emission of different clones of yeast treated with 0 nM, 0.5 nM or 10 nM 17h-estradiol and induced with 10 AM CuSO4. (B) Fluorescence emission of yeast cells induced or not induced with 10 AM CuSO4 and treated with different amounts of 17h-estradiol. With higher 17h-estradiol concentration the population is shifted to higher fluorescence emission.
response function [38], see Eq. (2). This function contains four parameters which describe the dose response behavior. The meaning of each parameter has been briefly described in the materials and methods section. In Fig. 6 the four parameters characterizing the logistic dose functions obtained with h-galactosidase as a reporter over a time range of approximately three years are plotted versus the number of experiments. These plots show accuracy and reproducibility. The yeast estrogen screen employing h-galactosidase as a reporter gene is very sensitive. The detection limit of the assay was defined as the mean value of the blank plus the three fold standard deviation of the blank. As a blank the pure
V. Beck et al. / J. Biochem. Biophys. Methods 64 (2005) 19–37
29
(A)
Miller units
100000
10000
Parameter a Parameter b
1000
100 0
20
40
60
80
100
120
Number of experiment
(B) 1,8e-9
0 -1
1,4e-9 -2
1,2e-9 1,0e-9
-3
8,0e-10
-4
6,0e-10
Parameter d
Parameter c [M]
1,6e-9
Parameter c Parameter d
-5
4,0e-10 -6
2,0e-10 0,0
-7 0
20
40
60
80
100
120
Number of experiment Fig. 6. Parameters of the logistic dose response curves obtained over a time frame of approximately three years in 127 experiments. (A) Parameter a is the baseline, b is the plateau of the curve, (B) c is the potency and d is the transition width.
solvent (DMSO) was taken and for 17h-estradiol a detection limit of 0.1 nM could be determined [8]. The sensitivity of the newly developed transactivation assay determined by analysis of various concentrations of 17h-estradiol was in the same range as for the conventional yeast estrogen screen (Figs. 7 and 8). The same low amounts of the standard substance can be detected. The potency of 17h-estradiol, which is the concentration necessary to reach half maximal transactivation, is also in the same range for both test systems. The performance of the estrogen screen using GFP as a reporter was compared with the performance of the established test system used in our lab for routine analysis. The aim of the study was to show that the newly developed yeast screen worked at least as well as the established routine estrogen screen. Various concentrations of fourteen different compounds belonging to different chemical classes (17h-estradiol, estrone,
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V. Beck et al. / J. Biochem. Biophys. Methods 64 (2005) 19–37
(A)
Relative Response [%]
250
200
17β-Estradiol Estrone Estriol 17α-Ethinylestradiol 17α-Estradiol Diethylstilbestrol Mestranol
150
100
50
0 1e-14 1e-13 1e-12 1e-11 1e-10 1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
Concentration [M]
(B) 250
Relative Response [%]
200
17β-Estradiol Coumestrol Daidzein Formononetin Genistein Biochanin A 4-Nonylphenol Zearalenone
150
100
50
0 1e-11 1e-10
1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
1e-3
Concentration [M] Fig. 7. Logistic dose response curves for various estrogenically active substances obtained in the yeast estrogen screen with h-galactosidase as a reporter. The response was related to E2. (A) shows the curves of various natural and synthetic estrogens (B) shows various phytoestrogens, the xenoestrogen 4-nonylphenol and the fungal estrogen zearalenone.
estriol, 17a-estradiol, 17a-ethinylestradiol, mestranol, diethylstilbestrol, zearalenone, 4nonylphenol, coumestrol, genistein, daidzein, formononetin and biochanin A) were analyzed with both test systems and logistic dose response curves were generated (Figs. 7 and 8). Potencies of the substances obtained in the tests were very similar using either the conventional yeast assay with h-galactosidase or the newly developed assay with GFP. Most of the potencies were also in accordance to values found in previous studies
V. Beck et al. / J. Biochem. Biophys. Methods 64 (2005) 19–37
31
(A) 140
Relative Response [%]
120
17β-Estradiol Estrone Estriol Mestranol 17α-Estradiol 17α-Ethinylestradiol Diethylstilbestrol
100 80 60 40 20 0 1e-14 1e-13 1e-12 1e-11 1e-10 1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
Concentration [M]
(B) 140
Relative Response [%]
120
17β-Estradiol Biochanin A Daidzein Formononetin Genistein Coumestrol 4-Nonylphenol Zearalenone
100 80 60 40 20 0 1e-11 1e-10
1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
1e-3
Concentration [M] Fig. 8. Logistic dose response curves for various estrogenically active substances obtained in the yeast estrogen screen with GFP as a reporter. The response was related to E2. (A) shows the curves of various natural and synthetic estrogens (B) shows various phytoestrogens, the xenoestrogen 4-nonylphenol and the fungal estrogen zearalenone.
[8]. To show the good agreement of the values obtained in the different assay systems the potency of the h-gal assay was plotted against the potency of the GFP-assay (Fig. 9). Most of the values are within the dashed lines y = 3x and 3y = x which surround the straight line y = x. This means that the potencies in both test systems are more or less identical. Values outside the dashed lines are more than three times higher or lower in one assay system than in the other. With these results it could be shown that the
32
V. Beck et al. / J. Biochem. Biophys. Methods 64 (2005) 19–37 1e-4
17β-Estradiol Estrone Estriol 4-Nonylphenol Coumestrol Biochanin A Daidzein Genistein Formononetin Mestranol Diethylstilbestrol Zearalenon 17α-Estradiol 17α-Ethinylestradiol
1e-5
Potency GFP-assay
1e-6
1e-7
1e-8
1e-9
1e-10
1e-11 1e-11
1e-10
1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
Potency β-Gal-assay Fig. 9. Comparison of potencies measured with test systems using h-gal or GFP as reporter. Potencies obtained in the h-gal yeast assay are plotted on the abscissa and potencies obtained in the GFP yeast assay were plotted on the ordinate. The dashed lines x = 3y and 3x = y are also shown. Symbols between the lines designate similar estrogenic activity in both test systems. Values outside the lines show potencies that are at least three times higher or lower in one assay than in the other.
applicable concentration range and the sensitivity are the same for both test systems and that the potencies of various substances are reproducible in the yeast transactivation assay, independent of the reporter plasmid used. In order to make the yeast estrogen screen more suitable for large sample sizes and fast analysis of estrogenic activities the test was down-scaled to be performed in microplates. The same concentrations of inductor substance and of 17h-estradiol standards were used as established for the larger scale of the test. Fluorescence measurements were performed on a microplate fluorescence reader after incubation for four, six and 24 h without any further sample treatment. Various incubation times for the yeast estrogen screen in microplates were tested. It was found that fluorescence measurements after six hours of incubation led to results that could be better fitted to a logistic dose response curve than those obtained after an incubation time of only four hours. Longer incubation times up to 24 h did not improve the quality of the resulting logistic dose response curve fits. Logistic dose response curves obtained in microplates are shown in Fig. 10. The incubation time of six hours did not alter the overall time needed for the assay compared to the h-gal yeast estrogen screen because the timeconsuming steps of cell disintegration, determination of h-gal activity and protein concentration after an incubation time of four hours could be omitted when GFP was used as a reporter gene. A comparison of potencies determined by the microplate assay and the
V. Beck et al. / J. Biochem. Biophys. Methods 64 (2005) 19–37
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(A) 120
Relative Response [%]
100
17β-Estradiol Estrone Estriol Mestranol 17α-Ethinylestradiol 17α-Estradiol Diethylstilbestrol
80
60
40
20
0 1e-15 1e-141e-13 1e-121e-11 1e-10 1e-9 1e-8 1e-7 1e-6 1e-5 1e-4
Concentration [M]
(B) 120
Relative Response [%]
100
17β-Estradiol Biochanin A Daidzein Formononetin Genistein Coumestrol 4-Nonylphenol Zearalenone
80
60
40
20
0 1e-11
1e-10
1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
1e-3
Concentration [M] Fig. 10. Logistic dose response curves for various estrogenically active substances obtained in microplates. (A) shows the curves of various natural and synthetic estrogens, (B) shows various phytoestrogens, the xenoestrogen 4-nonylphenol and the fungal estrogen zearalenone.
conventional protocol in tubes is shown in Fig. 11. Potencies of the logistic dose response curves of various compounds were determined and most of the values were in the same range as also found in the larger scale of the assay. However, the potencies of the estrogens 17a-estradiol, 17a-ethinylestradiol and mestranol obtained in the microplate assay were about two orders of magnitude higher than those obtained in the larger scale of the assay.
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V. Beck et al. / J. Biochem. Biophys. Methods 64 (2005) 19–37 1e-4
17β-Estradiol Estrone Estriol 4-Nonylphenol Coumestrol Biochanin A Daidzein Genistein Formononetin Mestranol Diethylstilbestrol Zearalenon 17α-Estradiol 17α-Ethinylestradiol
1e-5
Potency GFP-assay
1e-6
1e-7
1e-8
1e-9
1e-10
1e-11 1e-11
1e-10
1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
Potency micro-YES Fig. 11. Comparison of potencies measured with micro plate system and large scale system using GFP as reporter. Potencies obtained in microplate assay are plotted on the abscissa and potencies obtained large scale assay were plotted on the ordinate. The dashed lines x = 3y and 3x = y are also shown. Symbols between the lines designate similar estrogenic activity in both test systems. Values outside the lines show potencies that are at least three times higher or lower in one assay than in the other.
This indicates that lower concentrations of these substances can be detected in the microplate scale. Most of the potencies determined in the yeast assay in microplate scale are in accordance to the values obtained by other investigators using a similar test system [39]. The potency for the phytoestrogen coumestrol obtained in our newly developed assay was about one order of magnitude higher and the potencies for 17a-estradiol, 17aethinylestradiol and mestranol were about two orders of magnitude lower than those determined by Bovee et al. [39]. It is evident that the test system is more sensitive for these substances. We could also determine a potency value for the isoflavone daidzein, whereas Bovee et al. [39] did not find any response of ERa-expressing yeast to this compound. The high reproducibility of the microplate assay could be shown by performing the yeast estrogen screen with the same samples on different days. The parameters of the logistic dose response curves of the 17h-estradiol standard after six hours of incubation resulted in similar values on each day (Fig. 12). All parameters were in a quite narrow range in all assays performed. Parameter b shows the greatest variation of all parameters which might be due to the different age of the yeast culture used for inoculation of the over night culture used for the test. Although cells are freshly grown over night before each test, there are small variations in the number of fluorescing cells. The parameter a which equals the baseline of the curve, however, is not higher. This means that the correlation between the baseline and the plateau of the curve is higher when fresh yeast is used for inoculation
V. Beck et al. / J. Biochem. Biophys. Methods 64 (2005) 19–37
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100
26 24
Parameter b [fluorescence units]
Parameter a [fluorescence units]
(A) 90
22 80
20
70
18 16
60
14 50
12 10
40 0
2
4
6
8
10
12
14
16
18
Parameter a Parameter b
20
Number of experiment
(B) 0
7,0e-10
-1 5,0e-10 -2
4,0e-10
Parameter d
Parameter c [M]
6,0e-10
Parameter c Parameter d
3,0e-10 -3 2,0e-10 0
2
4
6
8
10
12
14
16
18
20
Number of experiment Fig. 12. Parameters of the logistic dose response curves of different batches after an incubation time of six hours obtained in a GFP yeast screen in microplates. (A) shows parameter a (baseline) and b (plateau of the curve), (B) shows parameter c (potency) and d (transition width).
of the over night culture and thus the range within which estrogenic activity can be detected is higher. Parameters c and d are quite constant over the time. Small variations are due to the biological nature of the test. The absolute range between baseline and plateau of the logistic dose response curve is higher in the yeast assay using h-gal as a reporter than in the GFP-microplate-assay. Nevertheless, good fits to the logistic dose response function can be obtained in the small scale assay.
4. Conclusion It could be shown that GFP functions as a reporter gene for the yeast estrogen screen and that the use of GFP as a reporter is a useful tool to make the assay procedure faster and
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easier. As there are less pipeting steps than in the conventional yeast estrogen screen with h-gal as a reporter gene, there are also less sources of error. In a routine analysis procedure the newly developed GFP-yeast assay can be used when high sample throughput in short time is required. Many microplates can be incubated and measured almost simultaneously and on each plate the equivalent estrogenic activity of at least five samples can be analyzed. As the yeast suspension volume per well is only 100 Al compared to 5 ml in the h-gal yeast assay, the GFP-assay is much cheaper regarding media requirements. As the potencies of all of the tested substances were in accordance to the conventional yeast screen, we concluded that the newly developed transactivation assay performed well enough to substitute the conventional h-gal transactivation assay.
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