Modulation of Estrogen Receptor-Dependent Reporter Construct ...

TOXICOLOGICAL SCIENCES 70, 193–201 (2002) Copyright © 2002 by the Society of Toxicology

Modulation of Estrogen Receptor-Dependent Reporter Construct Activation and G 0/G 1–S-Phase Transition by Polycyclic Aromatic Hydrocarbons in Human Breast Carcinoma MCF-7 Cells Jan Vondra´cˇek,* ,† ,1 Alois Kozubı´k,* and Miroslav Machala† *Laboratory of Cytokinetics, Institute of Biophysics, Kra´lovopolska´ 135, 612 65 Brno, Czech Republic; and †Department of Chemistry and Toxicology, Veterinary Research Institute, 621 32 Brno, Czech Republic Received May 31, 2002; accepted August 12, 2002

It has been suggested that the estrogenicity of PAHs could contribute to their carcinogenic effects via increased tissue-specific cell proliferation. Both benzo[a]pyrene (BaP) and benz[a]anthracene (BaA) are known to weakly activate estrogen receptor (ER)dependent reporter constructs. In this study, several other PAHs, including fluorene, fluoranthene, pyrene, chrysene, phenanthrene and anthracene, were found to act as very weak inducers of ER-mediated activity in the MCF-7 cell line stably transfected with a luciferase reporter gene. The effects of PAHs were timedependent and they were not completely inhibited by antiestrogen ICI 182,780. In addition, BaP and BaA, as well as weakly estrogenic fluoranthene, significantly potentiated the maximum ERmediated activity of 17␤-estradiol. Therefore, the effects of inhibitors of several types of protein kinases known to activate ER␣ in a ligand-independent manner were investigated. However, neither inhibitors nor inducers of extracellular signal-regulated kinases 1 and 2 (ERK1/2), phosphatidylinositol-3 kinase, protein kinase C, c-Src, or protein kinase A modified ER-mediated activity in this model. Neither estradiol nor BaA activated ERK1/2, two kinases suggested to play significant roles in ER signaling, suggesting that another kinase is involved in the observed phosphorylation of ER␣. Similar to 17␤-estradiol, BaA stimulated G 0/G 1–S-phase transition in MCF-7 cells, which was fully suppressed by ICI 182,780. In conclusion, some PAHs can potentiate 17␤-estradiolinduced ER activation and stimulate cell cycle entry in vitro. However, their exact mode(s) of action and whether this phenomenon is of in vivo relevance remains to be elucidated. Key Words: PAHs; MAPkinases; estrogenicity; cell cycle; ER␣.

A number of polycyclic aromatic hydrocarbons (PAHs), a class including diverse organic pollutants, are known or suspected carcinogens that have been reported to possess tumorinitiating and/or tumor-promoting properties (WHO, 1998). Although the vast majority of studies on PAHs have concentrated on the action of DNA-reactive PAH metabolites as initiating agents, increasing attention has also been paid in recent years to the nongenotoxic modes of PAH action, such as 1 To whom correspondence should be addressed. Fax: ⫹420-541211293. E-mail: [email protected].

the activation of aryl hydrocarbon receptor (AhR; Machala et al., 2001b; Piskorska-Pliszczynska et al., 1986), alteration of pathways regulating cell proliferation and survival (Tannheimer et al., 1998), inhibition of intercellular communication and/or activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in hepatic “stem-like” epithelial cells (Blaha et al., 2002; Rummel et al., 1999), or an increase in intracellular Ca 2⫹ in mammary epithelial cells (Tannheimer et al., 1999). PAHs have been reported to possess both estrogenic and antiestrogenic properties in various experimental settings. Today, PAHs are regarded mostly as antiestrogens, principally due to their ability to activate the Ah receptor, which leads to suppression of estrogen response element (ERE)-controlled gene expression via AhR-estrogen receptor (ER) cross-talk, increase in estradiol metabolism, and/or simultaneous downregulation of ER␣ levels (reviewed in Safe, 2001). However, recently, several PAHs, including benzo[a]pyrene (BaP) and benz[a]anthracene (BaA), or their oxygenated derivatives, have been shown to act as estrogenic compounds in ERregulated reporter gene assays (Charles et al., 2000; Fertuck et al., 2001b; Machala et al., 2001a). The in vivo estrogenicity of PAHs remains controversial; while some early studies suggested weak estrogenicity of 3,9-dihydroxy-7,12-dimethylbenz[a]anthracene in a rat uterotrophic assay (Morreal et al., 1979), others have found no evidence of in vivo estrogenicity of BaP and its metabolites (Fertuck et al., 2001b). It has been suggested that estrogenicity of PAHs is potentially the nongenotoxic component of their carcinogenic effects via increased organ-specific cell proliferation (Santodonato, 1997). The stimulation of cell cycle progression and cellular proliferation is a process that is likely to be associated with nongenotoxic, chemical-induced carcinogenesis (Foster, 1997). A number of xenoestrogenic compounds have been shown to act as effective mitogens in ER␣-expressing cells such as MCF-7 cells (Soto et al., 1995). Although it has been generally accepted that estrogen-mediated gene regulation is responsible for hormone-dependent cell proliferation, the exact mechanisms of cell cycle control by estrogens are still not fully understood (reviewed in Foster et al., 2001). Both estrogens

193

194

´ Cˇ EK, KOZUBI´K, AND MACHALA VONDRA

and peptide growth factors modulate levels, cellular redistribution, and activities of key cell-cycle regulators (reviewed in Pestell et al., 1999). Moreover, several rapid, so-called “nongenomic effects” of estrogens have been reported, including activation of two mitogen-activated protein kinases, ERK1/2, which participate in cell signaling associated with the induction of proliferation. However, while some authors have found a rapid and transient ERK1/2 activation within several minutes following estradiol addition (Improta-Brears et al., 1999; Migliaccio et al., 1996), others have not observed such response in breast cancer cells (Caristi et al., 2001; Joel et al., 1998; Lobenhofer and Marks, 2000). The cross-talk between peptide growth factor receptors and ER␣ can be even more complex, since ER␣ may be activated in a ligand-independent manner by a number of mechanisms, among which regulation of ER␣ phosphorylation by growth factors plays a significant role (reviewed in Cenni and Picard, 1999; Kato et al., 2000). An example thereof is the phosphorylation of ER␣ at serine 118 by activated ERK1/2. The phosphorylation of this serine residue has been shown to induce estrogen-like responses, such as proliferation of mammary and uterine epithelial cells and other estrogen-dependent cells and tissues (Arnold et al., 1995; Kato et al., 1995). Taken together, the potential role of PAHs in regulation of ER activity, as well as its impact on cell proliferation, still remain unclear. Both estrogens and growth factors employ some similar pathways regulating cellular proliferation. ERK1/2 rank with the major components of pathways controlling cell proliferation, differentiation, and apoptosis, and their activation was suggested as playing a role in estrogen-induced mitogenesis. The present study investigated the effects of PAHs on ER-dependent reporter construct activation and ERK1/2 activation in human breast carcinoma MCF-7 cells with respect to modulation of cell cycle progression. The aim was to define a mode of action that could potentially contribute to carcinogenic effects of PAHs in estrogen-dependent tissues. MATERIALS AND METHODS Cells and reagents. Human breast carcinoma MCF-7 cells (American Type Culture Collection, Rockville, MD) and their reporter gene variant MVLN cells (obtained from Dr. Michel Pons, Montpellier, France) were grown in a 1:1 mixture of Dulbecco’s minimal essential medium and Ham’s F-12 nutrient mixture (DMEM/F12, Sigma, St. Louis, MO) without phenol red, supplemented with 1 mM sodium pyruvate and 5% heat-inactivated fetal bovine serum (Sigma). H-89, forskolin, 8-bromocyclic adenosine monophosphate (Br-cAMP), phorbol 12-myristate 13-acetate (TPA), human recombinant epidermal growth factor (EGF), and 17␤-estradiol were purchased from Sigma. U0126, LY294002, rabbit polyclonal antiphospho-ERK1/2, rabbit polyclonal anti-ERK1/2, horseradish peroxidase-conjugated antirabbit IgG and antimurine IgG were from Cell Signaling Technology (Beverly, MA). ICI 182,780 was from Tocris (Bristol, UK). Murine monoclonal antihuman ER␣ antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). GF109203X, AG1478, and PP2 were from Alexis Biochemicals (San Diego, CA). Fluorene (CAS No. 86-73-7, purity 99.9%), anthracene (CAS No. 12012-7, purity 99.9%), phenanthrene (CAS No 85-01-8, purity 99.9%), fluoranthene (CAS No. 206-44-0, purity 99.9%), pyrene (CAS No. 129-00-0, purity

99.9%), benz[a]anthracene (CAS No. 86-73-7, purity 99.9%), chrysene (CAS No. 218-01-9, purity 99.9%), benzo[b]fluoranthene (CAS No. 205-99-2, purity 99.9%), benzo[k]fluoranthene (CAS No. 207-08-9, purity 99.9%), benzo[a]pyrene (CAS No. 50-32-8, purity 99.9%), indeno[1,2,3-c,d]pyrene (CAS No. 193-39-5, purity 99.9%), dibenz[a,h]anthracene (CAS No. 53-70-3, purity 99.9%), and benzo[ghi]perylene (CAS No. 191-24-2, purity 99.9%) were supplied by Ehrenstorfer (Augsburg, Germany). Reporter gene assay. MVLN cells stably transfected with the p-Vit-tk-Luc plasmid (Pons et al., 1990) were used for the detection of ER-mediated activity. The cell line was chosen due both to its consistence of response to 17␤-estradiol and its sensitivity to various types of phytoestrogens and xenoestrogens, being comparable to other established estrogenicity bioassays such as E-SCREEN (Guttendorf and Westendorf, 2001). The assays were run in 96-well cell culture plates. Briefly, 24 h after seeding, the medium was replaced with a medium supplemented with dextran/charcoal-treated serum for another 24 h. The cells were then dosed either with the tested compounds or with 17␤-estradiol calibration standards, prepared in dimethylsulfoxide (DMSO), and diluted with a serum-free medium, and were then incubated for a given time period (6 or 24 h). Maximum concentrations of DMSO did not exceed 0.1% (v/v). Cytotoxicity of PAHs was not observed at concentrations up to 20 ␮M (higher concentrations were not used because of a limited solubility of some compounds) as verified by MTT assay. In experiments where various types of inhibitors were used, these were applied to cells 90 min before the addition of the studied compounds. The concentrations of both effectors and inhibitors were based on available data. After exposure, the medium was removed, cells were washed and lysed with a low salt lysis buffer (10 mM Tris, 2 mM DTT, 2 mM 1,2-diamin cyclic hexane-N,N,N’,N’-tetraacetic acid, pH 7.8), and the plates were frozen at – 80°C. Luciferase activity was then measured in the microplate luminometer Luminoscan (Labsystems, Turku, Finland) using the Luciferase Monitoring Kit (Labsystems). Western blot analyses. MCF-7 cells were serum-starved for 24 h and then treated with selected compounds for the time indicated. After the incubation, the cells were washed with PBS and lysed with the sodium dodecyl sulfate (SDS) lysis buffer containing protease and phosphatase inhibitors (10% SDS, 1 mM phenylmethylsulfonyl fluoride, 10 nM aprotinin, 1 ␮M leupeptin, 1 ␮M antipain, 100 ␮M Na 3VO 4, and 5 mM NaF). Thirty ␮g of total protein per sample was separated on 12% polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. Activated ERK1/2 was detected with a phospho-specific polyclonal antibody and compared with total ERK1/2 expression, detected with a polyclonal antibody directed against a different epitope of ERK1/2. After incubation with primary and secondary antibodies, detection was performed with the ECLPlus Western blotting detection system (Amersham Pharmacia Biotech, Little Chalfont, UK). ER␣ phosphorylation was detected using the method described by Joel et al. (1998). Briefly, thirty micrograms of total protein per sample was separated on a 7% polyacrylamide gel until 35 kDa standard run off the gel, and transferred onto a polyvinylidene difluoride membrane, probed with antihuman ER␣ murine monoclonal antibody, and incubated with a secondary antibody conjugated with horseradish peroxidase. The acrylamide/bis-acrylamide ratio was changed to 99:1 in order to improve the separation of phosphorylated and unphosphorylated forms of ER␣. The secondary antibody was detected using the ECLPlus. A Model GS-670 Imaging Densitometer with Molecular Analyst Software (Bio-Rad Laboratories, Hercules, CA) was used to determine the relative amounts of total ER␣. Cell cycle analysis. MCF-7 cells were seeded in 12-well plates at an initial concentration of 24,000 cells per well. Cells were allowed to attach for 24 h, synchronized by serum withdrawal for 72 h, and treated with estradiol, BaA, or combinations thereof with antiestrogen ICI 182,780. After a 24-h incubation, cells were harvested, washed once with PBS, and then fixed in 5 ml of 70% ethanol at 4°C. Fixed cells were washed once with PBS and resuspended in 0.5 ml of Vindelov solution (1M Tris-HCl, pH 8.0; 0.1% Triton X-100, v/v; 10 mM NaCl; propidium iodide 50 ␮g/ml; RNase A 50 Kunitz units/ml) and incubated at 37°C for 30 min (Vindelov, 1977). Cells were then analyzed on

195

ESTROGENIC EFFECTS OF PAHs IN MCF-7 CELLS FACSCalibur, using a 488-nm (15 mW) air cooled argon-ion laser for propidium iodide excitation, and CELLQuest™ software for data acquisition (Becton Dickinson, San Jose, CA). A minimum of 15,000 events was collected per sample. Data were analyzed using ModFit LT version 2.0 software (Verity Software House, Topsham, ME). Statistics. Data were expressed as means ⫾ SD for at least three independent repeats and analyzed by the nonparametric Mann-Whitney U test or Kruskal-Wallis analysis of variance (ANOVA). A p value of less than 0.05 was considered significant.

RESULTS

Time dependence of an ER-mediated response. The luciferase reporter gene systems can reflect the high rate of PAH metabolism (Jones et al., 2000; Machala et al., 2001b). Thus, in the first series of experiments, we studied kinetics of luciferase induction by estradiol and selected PAHs: BaA, BaP, and fluoranthene. After 2 and 4 h, a small but significant increase in luciferase activity was observed, only after estradiol treatment (data not shown). After 6 h of incubation with BaA, luciferase activity was induced more than two-fold, which almost approached the effects of estradiol (2.5-fold induction), while BaP and fluoranthene induced lower luciferase activity. However, while the estradiol-induced luciferase activity continued to increase, that observed after PAH treatment decreased (BaA), remained constant (BaP), or declined back to control levels (fluoranthene). Because of this similarity to potencies of PAHs to transactivate AhR, which also depend on the rate of PAH metabolism, both 6-h and 24-h incubation periods were used in the following experiments. Relative potencies of 13 U.S. EPA priority PAHs (Callahan et al., 1979), other than highly volatile naphthalene, acenaphthalene, or acenaphthene, were determined using concentrations up to 20 ␮M (the concentration range was chosen because of limited solubility of some PAHs, such as chrysene and dibenzo[a,h]anthracene). The results are summarized in Table 1. Irrespective of the length of the incubation period, only BaA and BaP induced high levels of luciferase activity sufficient for the determination of 25% effective concentrations (EC25) and calculation of induction equivalency factors (IEFs) related to estradiol (see Table 1). The IEFs calculated after the 24-h exposure are up to one order of magnitude lower than those calculated from 6-h treatment data, probably due to metabolism of PAHs. An additional six PAHs (phenanthrene, anthracene, fluorene, fluoranthene, pyrene, and chrysene) induced a weak, but significant luciferase activity at high concentrations (5 ␮M and higher), following a 6-h incubation, while only chrysene induced a weak, but significant activity after 24 h. The remaining PAHs (indeno[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, benzo[k]fluoranthene, benzo[b]fluoranthene, and benzo[ghi]perylene) did not induce luciferase activity higher than the vehicle at either of the time points. Figure 1 shows the induction activities of concentration ranges of BaP, BaA, and a representative weak inducer (fluoranthene) after 6-h and 24-h treatments.

TABLE 1 Effects of Selected PAHs on ER-Dependent Reporter Construct Activity in MVLN Cells PAH

MW

6-h exposure

24-h exposure

Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benz[a]anthracene Chrysene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[a]pyrene Dibenzo[a,h]anthracene Indeno[1,2,3-cd]pyrene Benzo[ghi]perylene

166 178 178 202 202 228 228 252 252 252 278 276 276

wi wi wi wi wi 3.2 ⫻ 10 –6 wi ni ni 1.6 ⫻ 10 –6 ni ni ni

ni ni ni ni ni 7.9 ⫻ 10 –7 wi ni ni 2.6 ⫻ 10 –7 ni ni ni

Note. MVLN cells, human breast cancer MCF-7 cells permanently transfected with the p-Vit-tk-Luc gene construct. MW, molecular weight. The numbers represent induction equivalency factors (IEFs) calculated as the ratio between the 25% effective concentration (EC 25) of 17␤-estradiol and concentration of selected PAH inducing the same level of luciferase activity. Estradiol was used at a 1–1000 pM range; PAHs were applied at concentrations between 10 nM and 20 ␮M; ni, no induction of luciferase activity was observed up to 20 ␮M concentration of a given compound; wi, significant (p ⬍ 0.05), but too low induction of luciferase activity did not allow the calculation of IEFs. All the experiments were repeated 3 times in triplicate.

BaA, BaP and fluoranthene potentiate the effect of estradiol. Combined effects of PAHs (BaA, BaP and fluoranthene) and estradiol were investigated. BaP and BaA significantly potentiated the luciferase activity induced by estradiol (Fig. 2). This effect was observed using both the estradiol concentration, which induced the maximum luciferase activity (1 nM), and lower estradiol concentrations (data not shown). We also found that fluoranthene, which was only a weak inducer of ERmediated activity, potentiated the effects of estradiol to the same extent as BaP and BaA (Fig. 2). Effects of antiestrogen ICI 182,780 and modulators of protein kinase activities on ER-mediated induction of luciferase activity by BaP, BaA, fluoranthene and estradiol. As shown in Figure 3, the synthetic antiestrogen ICI 182,780 (Wakeling, 1995) inhibited both estradiol- and PAH-induced luciferase activities after 6 h of incubation. However, in contrast to the effect of estradiol, the cell lysates treated with PAHs still showed a significantly higher luciferase activity than the ICI 182,780-treated control. Apparently, a part of the induction capacity of PAHs was not inhibited by ICI 182,780. Following a 24-h incubation, effects of both PAHs and estradiol were completely suppressed by ICI 182,780, and the total level of luciferase activity was significantly lower than control values, presumably due to ER degradation induced by this antiestrogenic compound (data not shown) (MacGregor and Jordan, 1998). Therefore, we investigated whether inhibitors of several protein kinases known to phosphorylate and transactivate ER

196


FIG. 2. PAHs enhance ER-mediated transcription induced by estradiol. MVLN cells were treated with combinations of 1 nM estradiol and 10 ␮M BaA, 10 ␮M BaP, or 10 ␮M fluoranthene. Cell lysates were collected after 6-h incubation. The results of determination of luciferase activity are shown as % of maximum activity induced by 1 nM estradiol alone, and represent means ⫾ SD of 3 independent experiments run in triplicates. Open bars show luciferase activity after single-compound treatment, while the filled bars represent the activity after combined treatment with PAHs and estradiol. #Significant difference from estradiol-treated cells (p ⬍ 0.01).

cell line similar to estradiol or EGF, MCF-7 cells were serumstarved for 24 h and then incubated for 5 min to 6 h with estradiol, BaA, or EGF. Phosphorylation of ER␣ was detected as an electrophoretic up-shift of ER␣. As shown in Figure 5, estradiol induced a rapid and strong phosphorylation of ER␣ after 15 min of treatment and a significant portion of ER␣ FIG. 1. Induction of ER-mediated luciferase activity by estradiol, BaA, BaP, and fluoranthene in MVLN cells. MVLN cell lysates were harvested after 6 h (A) or 24 h (B) of stimulation with concentration ranges of 1 pM to 1 nM (estradiol), or 40 nM to 10 ␮M (BaA, BaP, and fluoranthene). The results of the determination of luciferase activity are shown as percentages of maximum activity induced by 1 nM of estradiol and are presented as means ⫾ SD, obtained in 3 independent experiments run in triplicate.

would inhibit PAH-induced ER-mediated transcription. The following compounds were used in the study: U0126, an inhibitor of ERK1/2 activation (Favata et al., 1998); LY294002, inhibitor of phosphatidylinositol-3-kinase (PI3K) (Vlahos et al., 1994); GF109203X, a general protein kinase C (PKC) inhibitor (Toullec et al., 1991); H-89, protein kinase A (PKA) inhibitor (Chijiwa et al., 1990); or PP2, a specific inhibitor of Src-family kinase (Hanke et al., 1996). As shown in Figure 4, none of the inhibitors significantly suppressed the luciferase activity induced by BaA, BaP or fluoranthene. Moreover, specific inducers of activity of the above-mentioned kinases (EGF, TPA, forskolin, and Br-cAMP) neither elicited significant luciferase activity nor potentiated the effect of estradiol in MVLN cells (Table 2). BaA induces a weak ER␣ phosphorylation. To determine whether PAHs induced phosphorylation of ER␣ in an MCF-7

FIG. 3. Inhibition of ER-mediated luciferase activity induced by estradiol and PAHs by synthetic antiestrogen—ICI 182,780. MVLN cells were pretreated for 30 min with 100 nM ICI182780 and then exposed to 1 nM estradiol, 10 ␮M BaA, 10 ␮M BaP, 10 ␮M fluoranthene, or DMSO as control. Cell lysates were collected after 6-h incubation. The results of determination of luciferase activity are shown as % of maximum activity induced by 1 nM estradiol and represent means ⫾ SD of 3 independent experiments run in triplicate. A slight decrease in luciferase activity associated with the degradation of ER induced by ICI 182,780 alone was observed. #Significant difference from control (p ⬍ 0.01). *Significant difference from control (p ⬍ 0.05).

ESTROGENIC EFFECTS OF PAHs IN MCF-7 CELLS

FIG. 4. Inhibitors of ERK1/2, PKC, PKA, PI3K or Src do not inhibit ER-mediated transcription induced by PAHs. MVLN cells were pretreated for 30 min with 10 ␮M U0126 (MEK1/2 inhibitor), 25 ␮M LY294002 (PI3K inhibitor), 5 ␮M PP2 (Src-family kinase inhibitor), 1 ␮M GF109203X (PKC inhibitor), or 10 ␮M H-89 (PKA inhibitor) and then exposed to 1 nM estradiol, 10 ␮M BaA, 10 ␮M BaP, or 10 ␮M fluoranthene. The results of determination of luciferase activity are shown as % of maximum activity induced by 1 nM estradiol and are presented as means ⫾ SD, obtained in 3 independent experiments run in triplicates.

remained phosphorylated up to 6 h. After 4 and 6 h of treatment, total ER␣ protein was decreased to 56.6 ⫾ 21.9 and 21.5 ⫾ 10.6% of control level, respectively, as determined by densitometry (expressed as mean ⫾ SD, n ⫽ 3). BaA appeared to induce phosphorylation of ER␣ after 15 min of treatment, but the amount of ER␣ phosphorylated was lower than in the case of estradiol-induced phosphorylation. Like estradiol, BaA lowered the levels of ER␣ protein after 4 and 6 h of treatment to 67.6 ⫾ 5.1 and 55.8 ⫾ 10.2% of control level, respectively, as determined by densitometry (expressed as mean ⫾ SD, n ⫽

197

FIG. 5. Time course of ER␣ phosphorylation induced by estradiol, EGF, and BaA. Serum-starved MCF-7 cells were treated for the time indicated with 1 nM estradiol, 100 ng/ml EGF, or 10 ␮M BaA. The cells were washed twice and harvested by addition of SDS lysis buffer with protease and phosphatase inhibitors. Cell lysates were resolved on SDS–PAGE and detected by Western blotting. The results are representative of 3 independent experiments.

3). EGF induced a rapid (within 5 min of treatment) phosphorylation of ER␣, but the maximum phosphorylation of ER␣ was only transient and a portion of the phosphorylated ER␣ remained low for up to 6 h. The time profile and extent of ER␣ phosphorylation corresponded with the time profile and the intensity of ERK1/2 phosphorylation after EGF treatment (see below). Estradiol and BaA do not induce a significant ERK1/2 phosphorylation. To determine whether PAHs, known to activate ER-mediated transcription, and estradiol modulate ERK1/2 activity in the MCF-7 model, the effects of estradiol and BaA on a time profile and intensity of ERK1/2 phosphorylation were studied, using a phosphospecific antibody. The results were compared with the effects of EGF. EGF induced a rapid and extensive phosphorylation of ERK1/2 within 5 min

TABLE 2 EGF, TPA, Br-cAMP and Forskolin Neither Induce LigandIndependent ER Activation Nor Enhance the Effect of Estradiol in MVLN Cells Treatment

Control

1 nM estradiol

DMSO Forskolin (1 ␮M) Br-cAMP (1 mM) EGF (100 ng/ml) TPA (100 nM)

38.4 ⫾ 0.8 42.7 ⫾ 1.3 47.5 ⫾ 4.9 40.6 ⫾ 2.8 40.1 ⫾ 8.7

100.1 ⫾ 2.3 100.2 ⫾ 15.6 105.2 ⫾ 9.3 115.4 ⫾ 8.6 104.8 ⫾ 6.7

Note. Cell lysates of MVLN cells were harvested after 6 h of stimulation with 1 ␮M forskolin, 1 mM Br-cAMP, 100 ng/ml EGF, or 100 nM TPA alone or in combination with 1 nM estradiol. The results of determination of luciferase activity are shown as % of maximum activity induced by 1 nM of estradiol, and are presented as means ⫾ SD, obtained in 3 independent experiments run in triplicate.

FIG. 6. Time course of ERK1/2 phosphorylation induced by EGF, estradiol, or BaA. Serum-starved MCF-7 cells were treated for the time indicated with 100 ng/ml EGF, 1 nM estradiol, or 10 ␮M BaA. The cells were washed twice and harvested by addition of SDS lysis buffer with protease and phosphatase inhibitors. Cell lysates were resolved on SDS-PAGE, transferred to PVDF membrane, and detected either with an antibody that recognizes only the dual-phosphorylated (Thr202/Tyr204) form of ERK1/2, or the total ERK1/2. The results are representative of 3 independent experiments.


198

TABLE 3 Effects of 17␤-Estradiol and Benz[a]anthracene on Percentage of MCF-7 Cells in S-Phase Treatment

% S-phase

Control ICI 182,780 Estradiol Estradiol ⫹ ICI 182,780 BaA BaA ⫹ ICI 182,780 Estradiol ⫹ BaA Estradiol ⫹ BaA ⫹ ICI 182,780

11.0 ⫾ 1.0 5.0 ⫾ 1.0 a,b 22.3 ⫾ 1.0 a 4.3 ⫾ 0.7 a,b 22.0 ⫾ 1.7 a 5.3 ⫾ 0.6 a,b 21.7 ⫾ 3.1 a 5.3 ⫾ 1.5 a,b

Note. Cells were synchronized and treated as described in Materials and Methods (72 h of serum deprivation ⫹ 24-h incubation with active compounds) with 1 nM 17␤-estradiol, 10 ␮M BaA, 100 ␮M ICI 182,780, or their combinations. Cells were then collected, stained with propidium iodide (PI), and analyzed by flow cytometry. The results are expressed as means ⫾ SD and represent 3 independent experiments run in duplicate. a Significantly different percentage of S-phase cells (p ⬍ 0.05) in comparison with untreated cells (Control). b Significantly different percentage of S-phase cells (p ⬍ 0.05) in comparison with cells treated only with estradiol, BaA, estradiol ⫹ BaA, or EGF, respectively.

of treatment, which declined after 15 min (Fig. 6). In contrast, both 1 nM estradiol and 10 ␮M BaA induced a weak, inconsistent ERK1/2 phosphorylation only after longer treatment with maximum observed after several hours (Fig. 6). However, this weak ERK1/2 phosphorylation was observed also after vehicle treatment (0.1% DMSO) or a simple manipulation with cell culture dishes (removal from CO 2 incubator and mixing of content) (data not shown). Therefore, the specificity of ERK1/2 phosphorylation was tested using inhibitors. This experiment (data not shown) demonstrated that the synthetic antiestrogen ICI 182,780 did not prevent induction of this weak ERK1/2 phosphorylation while it was blocked by both U0126 and AG1478 (a specific epidermal growth factor receptor [EGFR] inhibitor). S-phase induction. To compare the effects of estradiol and BaA, as the most potent estrogenic PAH on cell cycle progression, the cell cycle distribution was determined in MCF-7 cells. The cells were serum-starved for 72 h and then treated with BaA or estradiol or both for another 24 h. The serum deprivation is known to induce G 0/G 1 block in MCF-7 cells, from which they are released upon estradiol treatment (Foster et al., 2001). Both estradiol and BaA induced a significant increase in the percentage of cells in S-phase (see Table 3). However, the combination of BaA with estradiol did not lead to a further increase in the percentage of S-phase cells. ICI 182,780 blocked the effects of BaA, estradiol, or their combination, and it was found to inhibit cell cycle progression even below the level of control.

DISCUSSION

Metabolites of carcinogenic PAHs are potent initiation agents; however, relatively little is known about the promotional effects of PAHs and their metabolites, although these could play an important role in carcinogenesis. Some carcinogenic PAHs such as BaP have been shown to increase the incidence of tumors in estrogen-responsive rodent tissues (WHO, 1998), suggesting that they could affect ER-mediated signaling. A number of authors have reported that certain types of PAHs were antiestrogens (Arcaro et al., 1999; Chaloupka et al., 1992). Seven U.S. EPA priority PAHs, including BaP and BaA, were found to inhibit estradiol-dependent postconfluent cell growth in the MCF-7 focus assay (Arcaro et al., 1999). Their effects have been explained mainly by activation of AhR, a ligand-activated transcription factor that is considered to play the major role in carcinogenicity of dioxins and other planar polyaromatic compounds. Its effects on estrogen-dependent signaling are explained as an inhibitory AhR-ER␣ cross-talk at the cellular level. This cross-talk consists of at least three possible mechanisms, including activation of cytochrome P4501A1/2 enzymes leading to an increase in oxidative metabolism of estradiol; a complex gene promoter-specific inhibition of several estrogen-responsive genes; and proteasomedependent degradation of ER␣ (Safe, 2001). In contrast to the above reports, it has been shown that PAHs and/or their hydroxy- and oxy-derivatives can weakly activate ER-dependent reporter constructs in vitro (Charles et al., 2000; Fertuck et al., 2001b; Machala et al., 2001a). In the present study, we found that BaA and BaP can act as estrogenic compounds in the MCF-7 cell line stably transfected with the luciferase reporter gene, although at significantly higher concentrations than does estradiol. Metabolites of both BaP and BaA have been shown to act as estrogens (Charles et al., 2000; Fertuck et al., 2001b; Schneider et al., 1976). Moreover, a number of other PAHs, namely fluorene, fluoranthene, pyrene, chrysene, phenanthrene, and anthracene, acted as very weak inducers of ER-mediated activity in this study. The effects of PAHs were affected by their metabolism; while luciferase activity continued to rise following estradiol treatment of cells, the activity 24 h after addition of PAHs decreased or altogether disappeared when compared with ER-inducing potencies after a 6-h treatment (Fig. 1). This effect is apparently similar to AhR-mediated response, where metabolic clearance of PAHs, in contrast to highly persistent polyhalogenated aromatics, plays an important role in their time-dependent effects on this transcription factor (Jones et al., 2000; Machala et al., 2001b). Previously, no ER-mediated activity has been reported for the compounds found to be weakly active in MVLN cells (Fertuck et al., 2001a). However, this study used a chimeric Gal4 receptor-reporter system, which lacks ER activation function-1 (AF-1), while we employed a stably transfected reporter gene system under the control of endogenous ER␣. Another factor that might potentially contribute to the observed differences

ESTROGENIC EFFECTS OF PAHs IN MCF-7 CELLS

could have been the incubation period, as in the case of AhR-mediated activity of PAHs (Jones et al., 2000; Machala et al., 2001b). Nevertheless, our results do not exclude the possibility that the full-length ER expression is necessary for induction of these weak estrogenic effects in MVLN cells. The concentrations of PAHs required to observe a significant ER activation are relatively high, seemingly above their environmental levels. However, it has been shown recently that weak environmental estrogens can, at least in vitro, produce significant mixture effects when combined at concentrations below their NOECs (Silva et al., 2002). Therefore, as exposure to PAHs is always an example of a complex mixture exposure, mixture effects should be taken into consideration in their risk assessment. These facts have led us to explore in more detail the effects of PAHs on ER-dependent reporter construct and ER␣, which is the predominant form of ER in MCF-7 cells (Burow et al., 2000). ICI 182,780 belongs among “type II” antiestrogens that act as competitive inhibitors of ER and block the transport of newly synthesized ER into the nucleus, leading thus to its subsequent proteosomal degradation (MacGregor and Jordan, 1998). The current hypotheses of its action also suggest that although ICI 182,780-bound ER dimers still bind to EREs, ICI 182,780 effectively blocks the association with cofactors necessary for the formation of a transcriptionally active protein complex (MacGregor and Jordan, 1998). While ICI 182,780 fully inhibited estradiol-induced, ER-mediated transcriptional activity, we found a significantly higher luciferase activity in samples treated with a combination of PAHs and ICI 182,780 than in those treated with the antiestrogen alone. These results indicated that PAHs could perhaps still initiate the ER-mediated transcription by affecting the association of the ICI-ER␣ complex with cofactors. Moreover, estrogenic PAHs significantly enhanced the effects of estradiol, the cause of which could be related to different effects of these compounds on ER␣ (Fig. 6). ER␣ can be activated in a ligand-independent manner by a number of mechanisms, among which the regulation of ER␣ phosphorylation by growth factors plays a significant role (Cenni and Picard, 1999; Kato et al., 2000). The activation functions in ER␣, AF-1 and AF-2, can be activated by phosphorylation of the receptor at several, mostly serine, residues mediated by various protein kinases (reviewed in Cenni and Picard, 1999). However, from results gained after pretreatment of MVLN cells with specific inhibitors of ERK1/2 activation, it appears that PI3K, PKC, PKA, or Src-family kinases are not involved in effects of PAHs. Moreover, specific inducers of activity of the kinases mentioned above, such as EGF, TPA, forskolin, or Br-cAMP, neither elicited significant luciferase activity nor enhanced the effects of estradiol in MVLN cells model. Hence, ER␣ phosphorylation by these kinases is probably not involved in the observed effects. This finding does not exclude the possibility that PAHs could affect the interaction between ER␣ and one or more of its cofactors, or other signaling pathways leading to modulation of estro-

199

genic response. Both cell cycle regulators such as cyclin D1 (Cenni and Picard, 1999) and altered interaction with coactivators may transactivate ER␣ (Kato et al., 2000). Estradiol induces ER␣ phosphorylation, which modulates estradiol-induced transcriptional activation (Joel et al., 1998; Kato et al., 1995). The results of this study indicate that BaA could induce ER␣ phosphorylation in a similar manner as estradiol, albeit to a lower extent (Fig. 5). In accordance with earlier studies showing that phosphorylation of Ser 118 is involved in the regulation of transcriptional activity of AF-1 in response to EGF (Joel et al., 1998; Kato et al., 1995), EGF also induced transient ER␣ phosphorylation. The time profile of ERK1/2 phoshorylation after EGF treatment correlated with the time profile and the rate of ER␣ phosphorylation (Fig. 6). Several authors have suggested that estradiol, among other “nongenomic” effectors, can also induce a rapid ERK1/2 activation in MCF-7 cells (Improta-Brears et al., 1999; Migliaccio et al., 1996). On the other hand, results of other studies have not found a significant activation of ERK1/2 following estradiol treatment in this cell line (Caristi et al., 2001; Joel et al., 1998; Lobenhofer and Marks, 2000). It has been shown that ERK1/2 are not responsible for the ER␣ phosphorylation on Ser 118 in response to estradiol, and that another kinase or kinases must be involved in this process (Joel et al., 1998). In the present study, neither estradiol nor BaA induced ERK1/2 phosphorylation significantly (Fig. 6). Similar results were obtained also for BaP and fluoranthene (data not shown). The weak nonspecific effect observed may have been caused by cell manipulation (Caristi et al., 2001). Thus, together with the results reported in the previous paragraph, ERK1/2 do not seem to be involved in the effects of BaA. Estrogens are known to induce G 0/G 1-S transition in breast cancer cells and a number of cell cycle regulatory proteins have been shown to be affected by estradiol treatment during the early cell cycle events (reviewed in Foster et al., 2001). As it has been reported that PAHs may exert antiestrogenic effects by blocking estradiol-mediated postconfluent cell growth (Arcaro et al., 1999), the effect of BaA on cell cycle progression was investigated. Both estradiol and BaA were found to induce G 0/G 1 S-transition in a similar manner, although the combination of both compounds did not increase percentage of S-phase cells (Table 3). Their action was inhibited by synthetic antiestrogen ICI 182,780, which also blocked cell cycle progression more efficiently than serum starvation. These results seem to contradict published data on antiestrogenicity of AhR ligands (Arcaro et al., 1999; Wang et al., 1998). However, unlike the weakly estrogenic PAHs, persistent planar AhR inducers are not metabolized to compounds that would stimulate ER (Charles et al., 2000; Fertuck et al., 2001b) and may block the cell cycle progression through AhR-mediated mechanisms (Wang et al., 1998). Also, the effects of PAHs on subconfluent serum-deprived MCF-7 cell model used in the present study could have differed from the model using postconfluent, fourteen-day foci formation as an endpoint (Arcaro et al., 1999).


200

Taken together, a number of PAHs may act as weak estrogens in hormone-responsive human breast cancer cells. Importantly, the same compounds belong mostly among moderate and weak inducers (BaA, BaP, pyrene, phenanthrene, fluoranthene) or noninducers (fluorene, anthracene) of AhR-mediated activity (Machala et al., 2001b). Therefore, their respective antiestrogenic potency, mediated through the activation of AhR, could be counterbalanced by their direct ER-mediated activity. However, only BaA and BaP seem to have sufficient potency to act as weakly estrogenic compounds. Importantly, the ER-inducing PAHs, such as BaA, BaP, and fluoranthene, significantly potentiated the ER-mediated response to natural estrogen treatment. The most effective compound, BaA, as judged from the results of the assay of ER-dependent reporter construct activation, induced ER␣ phosphorylation in a similar manner to estradiol, although to a lesser extent. It was found that, like estradiol, BaA does not directly induce significant ERK1/2 phosphorylation, which, consequently, does not seem to play the key role either in phosphorylation of ER␣ or in a stimulation of cell-cycle progression observed after BaA treatment. Further studies are necessary to elucidate the exact mode(s) of action of PAHs, or to establish whether this phenomenon is of in vivo relevance and could play a promoting role in their tissue-specific carcinogenicity. ACKNOWLEDGMENTS The financial support from the Grant Agency of the Czech Republic (grant No. 525/01/D076 to J.V.) and the National Agency for Agricultural Research (grant No. QC0194 to M.M.) is gratefully acknowledged. The study was further supported by the Research Plan of the Academy of Sciences of the Czech Republic No. Z 5004920.

REFERENCES

stimulation of early G(1) progression in human breast cancer cells. Cancer Res. 61, 6360 – 6366. Cenni, B., and Picard, D. (1999). Ligand-independent activation of steroid receptors: New roles for old players. Trends Endocrinol. Metab. 10, 41– 46. Chaloupka, K., Krishnan, V., and Safe, S. (1992). Polynuclear aromatic hydrocarbon carcinogens as antiestrogens in MCF-7 human breast cancer cells: Role of the Ah receptor. Carcinogenesis 13, 2233–2239. Charles, G. D., Bartels, M. J., Zacharewski, T. R., Gollapudi, B. B., Freshour, N. L, and Carney, E. W. (2000). Activity of benzo[a]pyrene and its hydroxylated metabolites in an estrogen receptor-␣ reporter gene assay. Toxicol. Sci. 55, 320 –326. Chijiwa, T., Mishima, A., Hagiwara, M., Sano, M., Hayashi, K., Inoue, T., Naito, K., Toshioka, T., and Hidaka, H. (1990). Inhibition of forskolininduced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(pbromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J. Biol. Chem. 265, 5267–5272. Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J., Stradley, D. A., Feeser, W. S., Van Dyk, D. E., Pitts, W. J., Earl, R. A., Hobbs, F., Copeland, R. A., Magolda, R. L., Scherle, P. A., and Trzaskos, J. M. (1998). Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273, 18623–18632. Fertuck, K. C., Kumar, S., Sikka, H. C., Matthews, J. B., and Zacharewski, T. R. (2001a). Interaction of PAH-related compounds with the ␣ and ␤ isoforms of the estrogen receptor. Toxicol. Lett. 121, 167–177. Fertuck, K. C., Matthews, J. B., and Zacharewski, T. R. (2001b). Hydroxylated benzo[a]pyrene metabolites are responsible for in vitro estrogen receptormediated gene expression induced by benzo[a]pyrene, but do not elicit uterotrophic effects in vivo. Toxicol. Sci. 59, 231–240. Foster, J. R. (1997). The role of cell proliferation in chemically induced carcinogenesis. J. Comp. Pathol. 116, 113–144. Foster, J. S., Henley, D. C., Ahamed, S., and Wimalasena, J. (2001). Estrogens and cell-cycle regulation in breast cancer. Trends Endocrinol. Metab. 12, 320 –327. Guttendorf, B., and Westendorf, J. (2001). Comparison of an array of in vitro assays for the assessment of the estrogenic potential of natural and synthetic estrogens, phytoestrogens, and xenoestrogens. Toxicology 166, 79 – 89.

Arcaro, K. F., O’Keefe, P. W., Yang, Y., Clayton, W., and Gierthy, J. F. (1999). Antiestrogenicity of environmental polycyclic aromatic hydrocarbons in human breast cancer cells. Toxicology 133, 115–127.

Hanke, J. H., Gardner, J. P., Dow, R. L., Changelian, P. S., Brissette, W. H., Weringer, E. J., Pollok, B. A., and Connelly, P. A. (1996). Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T-cell activation. J. Biol. Chem. 271, 695–701.

Arnold, S. F., Obourn, J. D., Jaffe, H., and Notides, A. C. (1995). Phosphorylation of the human estrogen receptor by mitogen-activated protein kinase and casein kinase II: Consequence on DNA binding. J. Steroid Biochem. Mol. Biol. 55, 163–172.

Improta-Brears, T., Whorton, A. R., Codazzi, F., York, J. D., Meyer, T., and McDonnell, D. P. (1999). Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium. Proc. Natl. Acad. Sci. U.S.A. 96, 4686 – 4691.

Bla´ ha, L., Kapplova´ , P., Vondra´ cˇ ek, J., Upham, B., and Machala, M. (2002). Inhibition of gap-junctional intercellular communication by environmentally occurring polycyclic aromatic hydrocarbons. Toxicol. Sci. 65, 43–51.

Joel, P. B., Traish, A. M., and Lannigan, D. A. (1998). Estradiol-induced phosphorylation of serine 118 in the estrogen receptor is independent of p42/p44 mitogen-activated protein kinase. J. Biol. Chem. 273, 13317– 13323.

Burow, M. E., Weldon, C. B., Chiang, T. C., Tang, Y., Collins-Burow, B. M., Rolfe, K., Li, S., McLachlan, J. A., and Beckman, B. S. (2000). Differences in protein kinase C and estrogen receptor ␣, ␤ expression, and signaling correlate with apoptotic sensitivity of MCF-7 breast cancer cell variants. Int. J. Oncol. 16, 1179 –1187. Callahan, M. A., Slimak, M. W., Gabel, N. W., May, I. P., Flower, C. F., Freed, J. R., Jennings, P., DuPree, R. I., Whitmore, F. C., Maestri, B., Mabey, W. R., Holt, B. R., and Gould, C. (1979) Water-related environmental fate of 129 priority pollutants. In EPA-440/4 –79 – 029a and b, Vols. I and II. U.S. EPA, Springfield, VA. Caristi, S., Galera, J. L., Matarese, F., Imai, M., Caporali, S., Cancemi, M., Altucci, L., Cicatiello, L., Teti, D., Bresciani, F., and Weisz, A. (2001). Estrogens do not modify MAP kinase-dependent nuclear signaling during

Jones, J. M., Anderson, J. W., and Tukey, R. H. (2000). Using the metabolism of PAHs in a human cell line to characterize environmental samples. Environ. Toxicol. Pharmacol. 8, 119 –126. Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawashima, H., et al. (1995). Activation of the estrogen receptor through phosphorylation by mitogenactivated protein kinase. Science 270, 1491–1494. Kato, S., Masuhiro, Y., Watanabe, M., Kobayashi, Y., Takeyama, K. I., Endoh, H., and Yanagisawa, J. (2000). Molecular mechanism of a cross-talk between oestrogen and growth factor signalling pathways. Genes Cells 5, 593– 601. Lobenhofer, E. K., and Marks, J. R. (2000). Estrogen-induced mitogenesis of

ESTROGENIC EFFECTS OF PAHs IN MCF-7 CELLS MCF-7 cells does not require the induction of mitogen-activated protein kinase activity. J. Steroid Biochem. Mol. Biol. 75, 11–20. MacGregor, J. I., and Jordan, V. C. (1998). Basic guide to the mechanisms of antiestrogen action. Pharmacol. Rev. 50, 151–196. Machala, M., Ciganek, M., Bla´ ha, L., Minksova´ , K., and Vondra´ cˇ ek, J. (2001a). Aryl hydrocarbon receptor-mediated and estrogenic activities of oxygenated polycyclic aromatic hydrocarbons and azaarenes originally identified in extracts of river sediments. Environ. Toxicol. Chem. 20, 2736 – 2743. Machala, M., Vondra´ cˇ ek, J., Bla´ ha, L., Ciganek, M., and Necˇ a, J. V. (2001b). Aryl hydrocarbon receptor-mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using in vitro reporter gene assay. Mutat. Res. 497, 49 – 62. Migliaccio, A., Di Domenico, M., Castoria, G., de Falco, A., Bontempo, P., Nola, E., and Auricchio, F. (1996). Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. EMBO J. 15, 1292–1300. Morreal, C. E., Schneider, S. L., Sinha, D. K., and Bronstein, R. E. (1979). Estrogenic properties of 3,9-dihydroxy-7,12-dimethylbenz[a]anthracene in rats. J. Natl. Cancer Inst. 62, 1585–1588. Pestell, R. G., Albanese, C., Reutens, A. T., Segall, J. E., Lee, R. J., and Arnold, A. (1999). The cyclins and cyclin-dependent kinase inhibitors in hormonal regulation of proliferation and differentiation. Endocr. Rev. 20, 501–534. Piskorska-Pliszczynska, J., Keys, B., Safe, S., and Newman, M. S. (1986). The cytosolic receptor-binding affinities and AHH induction potencies of 29 polynuclear aromatic hydrocarbons. Toxicol. Lett. 34, 67–74. Pons, M., Gagne, D., Nicolas, J. C., and Mehtali, M. (1990). A new cellular model of response to estrogens: A bioluminescent test to characterize (anti)-estrogen molecules. Biotechniques 9, 450 – 459. Rummel, A. M., Trosko, J. E., Wilson, M. R., and Upham, B. L. (1999). Polycyclic aromatic hydrocarbons with bay-like regions inhibited gap junctional intercellular communication and stimulated MAPK activity. Toxicol. Sci. 49, 232–240. Safe, S. (2001). Molecular biology of the Ah receptor and its role in carcinogenesis. Toxicol. Lett. 120, 1–7. Santodonato, J. (1997). Review of the estrogenic and antiestrogenic activity of

201

polycyclic aromatic hydrocarbons: Relationship to carcinogenicity. Chemosphere 34, 835– 848. Schneider, S. L., Alks, V., Morreal, C. E., Sinha, D. K., and Dao, T. L. (1976). Estrogenic properties of 3,9-dihydroxybenz[a]anthracene, a potential metabolite of benz[a]anthracene. J. Natl. Cancer Inst. 57, 1351–1354. Silva, E., Rajapakse, N., and Kortenkamp, A. (2002). Something from “nothing”— eight weak estrogenic chemicals combined at concentrations below NOECs produce significant mixture effects. Environ. Sci. Technol. 36, 1751–1756. Soto, A. M., Sonnenschein, C., Chung, K. L., Fernandez, M. F., Olea, N., and Serrano, F. O. (1995). The E-SCREEN assay as a tool to identify estrogens: An update on estrogenic environmental pollutants. Environ. Health Perspect. 103(Suppl. 7), 113–122. Tannheimer, S. L., Ethier, S. P., Caldwell, K., and Burchiel, S. W. (1998). Benzo[a]pyrene- and TCDD-induced alterations in tyrosine phosphorylation and insulin-like growth factor signaling pathways in the MCF-10A human mammary epithelial cell line. Carcinogenesis 19, 1291–1297. Tannheimer, S. L., Lauer, F. T., Lane, J., and Burchiel, S. W. (1999). Factors influencing elevation of intracellular Ca2⫹ in the MCF-10A human mammary epithelial cell line by carcinogenic polycyclic aromatic hydrocarbons. Mol. Carcinog. 25, 48 –54. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet V., Boissin, P., Boursier, E., Loriolle, F., et al. (1991). The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266, 15771–15781. Vindelov, L. L. (1977). Flow microfluorimetric analysis of nuclear DNA in cells from solid tumors and cell suspensions. A new method for rapid isolation and staining of nuclei. Virchows Arch. B Cell Pathol. 24, 227–242. Vlahos, C. J., Matter, W. F., Hui, K. Y., and Brown, R. F. (1994). A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4- morpholinyl)-8-phenyl-4H1-benzopyran-4-one (LY294002). J. Biol. Chem. 269, 5241–5248. Wakeling, A. E. (1995). Use of pure antioestrogens to elucidate the mode of action of oestrogens. Biochem. Pharmacol. 49, 1545–1549. Wang, W., Smith, R., III, and Safe, S. (1998). Aryl hydrocarbon receptormediated antiestrogenicity in MCF-7 cells: Modulation of hormone-induced cell cycle enzymes. Arch. Biochem. Biophys. 356, 239 –248. WHO (1998). Selected Non-Heterocyclic Polycyclic Aromatic Hydrocarbons, Vol. 202. World Health Organization, Geneva.