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Aryl Hydrocarbon Receptor-Mediated Antiestrogenicity in MCF-7 Cells: Modulation of Hormone-Induced Cell Cycle Enzymes
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 356, No. 2, August 15, pp. 239 –248, 1998 Article No. BB980782
Aryl Hydrocarbon Receptor-Mediated Antiestrogenicity in MCF-7 Cells: Modulation of Hormone-Induced Cell Cycle Enzymes1 Weili Wang, Roger Smith, III, and Stephen Safe2 Department of Veterinary Physiology and Pharmacology and Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas 77843-4466
Received April 20, 1998, and in revised form May 23, 1998
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits 17b-estradiol (E2) mammary tumor growth in rodents and in MCF-7 human breast cancer cells; however, the cell cycle genes/proteins which are inhibited have not been determined. Initial studies showed that treatment of MCF-7 cells with 10 nM E2 significantly increased cyclin D1 (protein and mRNA), cdk2- and cdk4-dependent kinase activities, and hyperphosphorylation of retinoblastoma (RB) protein. In contrast to results of recent studies (M. D. Planas-Silva and R. A. Weinberg, 1997, Mol. Cell. Biol. 17, 4059 – 4069), E2 induced dissociation of both cdk2 and cdk4 proteins from the p21 protein complex and significantly increased cdk7-dependent kinase activity. Treatment of MCF-7 cells with E2 also induced cdc25A phosphatase protein, which was accompanied by increased cdk2 and cdk4 proteins containing unphosphorylated tyrosine residues. Although TCDD alone has minimal effects on cell cycle proteins/enzymes, several E2-induced responses were significantly inhibited in MCF-7 cells cotreated with E2 plus TCDD. For example, TCDD significantly inhibited E2-induced hyperphosphorylation of RB, cyclin D1 protein, and cdk2-, cdk4-, and cdk7-dependent kinase activities. Inhibition of E2-induced cdk4-dependent kinase activity by TCDD may be related to the parallel decrease of E2-induced cyclin D1 protein, and inhibition of induced cdk2- and cdk4-dependent kinase activities may be due to significantly increased p21 levels in cells cotreated with TCDD plus E2. These results demonstrate that the antiestrogenic activity of TCDD is due to downregula-
1 The financial assistance of the National Institutes of Health (CA-64081 and ES04176) and the Texas Agricultural Experiment Station is gratefully acknowledged. 2 To whom correspondence should be addressed. Fax: (409) 8624929. E-mail: [email protected].
0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
tion of several E2-induced cell cycle proteins/activities and this illustrates the complex cross talk between the aryl hydrocarbon and the E2 receptor signaling pathways. © 1998 Academic Press Key Words: estrogen; 2,3,7,8-tetrachlorodibenzo-pdioxin; cell cycle; cross-talk mechanisms.
Estrogenic hormones play an essential role in mammalian physiology (1); however, it has also been demonstrated that lifetime exposure to estrogens is a major risk factor for both breast and endometrial cancer in women (2– 4). Although the precise roles of 17b-estradiol (E2)3 and related estrogenic hormones in development of mammary cancer have not been established (5, 6), it is evident from both laboratory animal and in vitro cell culture studies that estrogen receptor (ER)mediated gene expression and growth factors are important components of the cancer process (7–12). Several studies have investigated the effects of E2, antiestrogens, and other inhibitors of mammary cancer cell growth and cell cycle enzymes in ER-positive MCF-7 or T47D human breast cancer cell lines (13–25). Foster and Wimalasena (13) reported that in growtharrested MCF-7 cells, 89.4% of the cells were in G0/G1, and only 6.6 and 4.0% were in S and G2/M phase, respectively. The antiestrogen ICI 182,780 maintained this distribution of cells in different phases of the cell cycle, whereas treatment with E2 decreased the per3 Abbreviations used: E2, 17b-estradiol; ER, estrogen receptor; RB, retinoblastoma; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AhR, aryl hydrocarbon receptor; DRE, dioxin-responsive element; i, inhibitory; DME, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; DCC, dicyclohexylcarbodiimide; FBS, fetal bovine serum; CAK, cdk-activating kinase; GST, glutathione S-transferase; ECL, enhanced chemiluminescence.
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centage of cells in G0/G1 (65.2%) and increased cells in S (20.6%) and G2/M (14.2%) phases. These changes in cell cycle distribution were accompanied by increased cdk2- and cdk4-associated kinase activities, increased phosphorylation of retinoblastoma (RB) protein, and increased cyclin D1 protein levels. E2 also decreased cdk-inhibitory activities associated with p27kip-1 and this was accompanied by lower p27kip-1 protein levels. Similar results were reported by Prall and co-workers (14), who utilized MCF-7 cells growth arrested by the antiestrogen ICI 182,780. Their results also showed that hormone-induced activation of cyclin E– cdk2 complexes resulted in decreased association with cdk inhibitors p27kip-1 and p21 and increased phosphorylation of cdk2 threonine-160. In tamoxifen growth-arrested cells, Planas-Silva and Weinberg (15) reported that treatment with E2 also resulted in redistribution of cdk inhibitor p21 from its association with cyclin E– cdk2 to cyclin D– cdk4. These studies exhibited some similarities and differences which may be due to differences in experimental methodologies and methods for arresting growth of MCF-7 cells. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a compound which binds with high affinity to the aryl hydrocarbon receptor (AhR), and several studies show that TCDD and related AhR agonists inhibit diverse E2-induced responses (reviewed in 26). The mechanisms of cross talk between AhR and ER signaling pathways have been investigated using cathepsin D and pS2 genes as models (27, 28). Inhibition of E2induced pS2 and cathepsin D gene expression by TCDD involves interaction of the nuclear AhR complex with GCGTG sequences (27, 28) containing the core binding motif of the dioxin-responsive element (DRE) which forms part of the genomic enhancer element required for AhR-mediated transactivation (29 –32). The inhibitory DRE (iDRE) core binding motifs in the pS2 and cathepsin D gene promoters interact with the nuclear AhR complex; however, enhanced gene expression is not observed, due to the absence of specific nucleotides which flank the core iDRE. AhR agonists also inhibit E2- and growth factor-induced proliferation of MCF-7 cells (33– 40) and formation and growth of mammary tumors in rodent models (41– 45). This study utilized TCDD as a probe for determining AhRmediated effects on key cell cycle proteins which are involved in growth-promoting activity of estrogens. In this study, E2 induced cyclin D1 protein, cdk2- and cdk4-dependent kinase activities, and RB phosphorylation in MCF-7 cells, and all of these responses were inhibited by TCDD. These interactive effects were also accompanied by rapid upregulation of p21 protein in MCF-7 cells treated with TCDD alone or with TCDD plus E2; induction of this cdk inhibitor, coupled with inhibition of E2-induced cell cycle enzyme activities, demonstrates that the antiestrogenic activity of AhR
agonists is associated with modulation of multiple cell cycle enzymes and proteins. MATERIALS AND METHODS Chemicals and antibodies. E2 and histone (type III-SS) were purchased from Sigma Co. (St. Louis, MO). TCDD was prepared in this laboratory and was .98% pure as determined by high-pressure liquid and gas chromatography. Cyclin D1, cyclin A, cyclin E, cyclin H, cdk2, cdk4, cdk7, p21, p27, p53, cdc 25A, phosphotyrosine (PY20) and RB antibodies, protein A–agarose beads, and GST-RB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). [g-32P]ATP (3000 Ci/mmol) was obtained from New England Nuclear (Boston, MA). All other chemicals and biochemicals were purchased from commercial sources and were either reagent or molecular grade. Cell culture maintenance and growth. MCF-7 human breast cancer cells were obtained from the American Type Culture Collection (ATCC) and maintained in DME/F12 medium without phenol red and supplemented with 5% fetal bovine serum plus 1% antibiotic/ antimycotic solution at 37°C. For experiments, cells were seeded into 100-mm petri dishes and grown until 70% confluence was reached. Cells were then synchronized in DME/F12 serum-free medium for 3 days; cells were then treated with DMSO (0.1% total volume), 10 nM E2, E2 plus 10 nM TCDD, and 10 nM TCDD alone for 6, 12, and 24 h, respectively. Cells were then harvested and different cell parameters were determined as described below. Preparation of whole-cell extracts. Cells, treated as described above, were washed once in ice-cold PBS and scraped into lysis buffer (50 nM Hepes (pH 7.5), 150 mM sodium chloride, 10% (v/v) glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 200 mM sodium orthovanadate, 10 mM pyrophosphate, and 100 mM NaF). Cells were incubated for 30 min and then centrifuged at 10,000g for 5 min. Supernatants were precleared by addition of 20 ml of protein A–agarose beads for 30 min followed by centrifugation for 5 min at 10,000g. The lysates used for both Western blot and kinase assays were stored at 280°C until required. All procedures were carried out at 4°C. Western immunoblot analysis and immunoprecipitation. Equal amounts of protein from cell lysates prepared as described above were separated by SDS–PAGE and then transferred to nitrocellulose membrane using an electroblotting apparatus overnight at 4°C. Membranes were blocked with TBS (10 mM Tris–HCl, pH 8.0; 150 mM sodium chloride) plus 5% milk (Blotto buffer) for 1 h and then incubated in primary antibody at 0.1 to 0.5 mg/ml in the Blotto buffer for 1 to 2 h at 20°C. Membranes were rinsed once and washed for 6 min (33) in TBS buffer. The secondary anti-mouse or anti-rabbit/ HRP (1:1000 –5000) was added to Blotto buffer and incubated for 1 h at 20°C. Membranes were washed as described above and incubated in NEN ECL reagents for 1 min; excess ECL reagent was removed by dabbing with a Kimwipe, and membranes were sealed in plastic wrap. Membranes were then exposed to ECL hyperfilm for visualization of immunoreactive bands. Protein levels were quantitated using a Sharp JX-330 densitometer and a Scanalytics Zero-D software package (Scanalytics, Billerica, MA). For p21 immunoprecipitation and immunodepletion, cell lysates (350 mg protein in 150 ml) were immunoprecipitated by three sequential immunoprecipitations with 0.3 mg p21 antibody and 9 ml of protein A–agarose beads. Beads were then combined, washed three times with lysis buffer, boiled for 5 min in 23 SDS sample buffer, loaded on SDS–PAGE, and then assayed by Western blot analysis for cyclin D1 to investigate the association between p21 and cyclin D1. The p21-immunodepleted supernatants were saved and equal volumes (30 ml) of supernatants were loaded onto 10% SDS–PAGE and followed by Western blot analysis for cdk2 and cdk4 to examine the association of p21 with
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ESTROGEN AND 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN IN THE CELL CYCLE cdk2 and cdk4. The procedure used for PY20 immunodepletion was similar to that described for p21; PY20-immunodepleted supernatants were used to quantitate levels of unphosphorylated-tyrosine cdk2 and cdk4 by Western blot analysis. Quantitation of immunoreactive proteins was determined by densitometry. Kinase assays. Cell lysates (350 mg for cdk2 and cdk4 kinase assays; 200 mg for cdk7 kinase assay), prepared as described above, were incubated with individual antibodies (300 to 500 ng/each) for 3 to 15 h at 4°C and then immunoprecipitated by incubation with protein A–agarose beads for 3 h at 4°C. Beads were washed with lysis buffer (33) and with kinase buffer (33) (50 mM Tris–Hepes, pH 7.5, 10 mM MgCl2). Kinase reaction was carried out in 30 ml of kinase buffer supplemented with 400 mg/ml histones (Sigma type III-SS) for cdk2 and cdk7 kinase assays or 0.3 mg GST-RB (sc-4112; Santa Cruz Biotechnology) for cdk4 kinase assay (10 mM ATP, 0.5 mM dithiothreitol, 0.5 mM EGTA, and 5 mCi [g-32P]ATP for 20 to 40 min at 30°C). Assays were stopped with 35 ml of 23 SDS–PAGE sample buffer and boiled for 5 min; samples were then loaded and separated on 10% SDS–PAGE. The 30-kDa phosphorylated histone and 50-kDa phosphorylated GST-RB products were readily observed and were visualized by autoradiography using Kodak film. Intensities of specific bands were quantitated by densitometry as described above. Flow cytometry analysis. Cells were seeded in six-well plates in DME/F12 medium supplemented with 2.5% DCC–FBS. Cells were synchronized in serum-free DME/F12 medium for 3 days. Cells were then treated with 10 nM E2, 10 nM E2 plus 10 nM TCDD, and 10 nM TCDD in serum-free medium for 12 and 24 h, respectively. Cells were washed once with PBS, harvested, resuspended in 0.5 ml of PBS, fixed by dropwise addition of 4.5 ml of ice-cold 70% ethanol, and stored overnight at 220°C. Fixed cells were then washed once and resuspended in a propidium iodide staining buffer, consisting of 0.1% (v/v) Triton X-100 (Sigma), 200 mg/ml DNase-free RNase A (Sigma), and 50 mg/ml propidium iodide (Sigma). Cells were incubated for 1 to 6 h at 20°C prior to analysis on a FACS Calibur instrument (Becton– Dickinson, San Jose, CA). Data were analyzed in ModFit LT for the Macintosh, version 2.0 (Verity Software House, Topsham, ME), using the diploid model, with visible G2M peak, for fresh/frozen cells. The coefficient of variation varied from 3.8 to 5.3% for all samples. Statistics. Results are expressed as means 6 SE for at least three independent (replicate) experiments for each treatment group. Statistical significance was determined by ANOVA and Student’s t test and the levels of probability are noted.
RESULTS
Effects of E2, TCDD, and TCDD plus E2 on cell cycle distribution of MCF-7 cells and cell cycle proteins. MCF-7 cells were maintained in DME/F12 medium containing 5% fetal bovine serum and cells were then cultured for 3 days in serum-free medium prior to treatment with E2, TCDD, or TCDD plus E2 for 6, 12, or 24 h. These experiments were all carried out in serum-free medium. Using this protocol, the maximal effects on cell cycle parameters were observed for 10 nM E2, and 10 nM TCDD was the optimal concentration for inhibiting E2-induced responses. The cell cycle distribution of MCF-7 cells was determined by flow cytometry; cells in S phase were not increased after treatment with E2 for 12 h; however, a significant increase in the percentage of cells in S phase and a decreased percentage in G0G1 were observed after treatment for 24 h (Table I). TCDD alone had minimal effects; however, in cells cotreated with TCDD plus E2,
TABLE I
Effects of 17b-Estradiol and TCDD on Cell Cycle Distribution of MCF-7 Human Breast Cancer Cellsa Cell cycle phase (%) Treatment (time, h)
G0/G1
S
G2/M
Control E2 (12) E2 1 TCDD (12) TCDD (12) E2 (24) E2 1 TCDD (24) TCDD (24)
89.9 6 2.1 87.7 6 2.1 87.2 6 0.2 89.1 6 0.8 75.1 6 0.6b 81.0 6 1.3c 90.8 6 0.6
4.9 6 1.6 6.0 6 1.4 7.9 6 0.7 6.7 6 0.8 23.4 6 1.7b 15.8 6 1.8d 5.2 6 0.5
5.2 6 0.6 4.4 6 0.7 4.9 6 0.5 4.2 6 0.2 1.5 6 1.2 3.2 6 0.7 4.0 6 0.9
a Cells were seeded in six-well plates at 3 3 105 cells/well in DME/F12 medium supplemented with 2.5% DCC–FBS. Cells were synchronized in serum-free DME/F12 medium (SFM) for 3 days. Cells were then treated with 10 nM E2, 10 nM E2 plus 10 nM TCDD, and 10 nM TCDD in SFM for 12 and 24 h, respectively. Cells were washed once and the assay was carried out as described under Materials and Methods. The cell cycle phase distribution in DMSOtreated (control) cells was determined after 24 h. Experiments were performed in triplicate and results are expressed as means 6 SE. b Significantly different (P , 0.001) from control cells at respective time points. c Significantly different (P , 0.04) from E2 24-h-treated cells at G0/G1 phase. d Significantly different (P , 0.001) from E2 24-h-treated cells at S phase.
there was significant inhibition of the hormone-induced increase in cells in S phase. The results summarized in Fig. 1 show that immunoreactive levels of cyclin E, cyclin A, cdk2, and cdk4 protein were not significantly changed in any of the treatment groups. E2 significantly induced phosphorylation of the active form of cdk2 (*cdk2, Fig. 1) as previously reported (14, 15). Cyclin D1 levels were decreased slightly in MCF-7 cells treated with TCDD alone, whereas E2 alone caused a rapid (within 6 h) and sustained 2.8- to 3-fold increase in cyclin D1 protein (Fig. 2). In cells cotreated with E2 plus TCDD, the hormone-induced response was significantly decreased after 12 or 24 h treatment with TCDD. The effects observed for E2 alone on this same set of cell cycle proteins were similar to those previously described (13), and suppression of E2-induced cyclin D1 by TCDD was comparable to the effects observed for the pure antiestrogen ICI 182,780 (13). Modulation of cdk4- and cdk2-dependent kinases, p21, p27, and p53. E2 alone caused a relatively rapid (within 6 h) 3.8-fold increase in cdk4-dependent kinase activity, which decreased to background levels after 24 h (Fig. 3). In contrast, cdk2-dependent kinase activity was induced more slowly and, after 24 h, a 2.4-fold increase was observed. TCDD alone did not affect cdk2or cdk4-dependent kinase activity; however, in cells cotreated with E2 plus TCDD, the hormone-induced activities were decreased at all time points. Treatment
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FIG. 1. Western blot analysis of effects of E2 and TCDD on cyclin E, cyclin A, cdk2, and cdk4 protein expression in MCF-7 cells. Cells were seeded into 100-mm petri dishes and synchronized for 3 days in serum-free DME/F12 medium. Cells were then treated with DMSO (0.1% v/v) as control (U), 10 nM E2, 10 nM E2 plus 10 nM TCDD, and 10 nM TCDD alone for 6, 12, and 24 h, respectively. Whole-cell lysates (100 mg), prepared as described under Materials and Methods, were used for the assay. The protein levels in the treated groups were not significantly changed compared to DMSO– control cells. However, the hyperphosphorylated form of cdk2 was significantly increased 2.4 6 0.3-fold (P , 0.05) with E2 treatment at 24 h compared to the control. TCDD only slightly blocked E2 effects at this time and not significantly. The Western blot assay was carried out as described under Materials and Methods.
of MCF-7 cells with E2 or TCDD had minimal effects on p27 cdk inhibitor protein levels; however, p27 protein levels were significantly elevated in cells cotreated with E2 1 TCDD (Fig. 4). E2 alone did not affect levels of p21 protein, whereas TCDD alone and in combination with E2 significantly elevated p21 levels with the highest increase (1.8- to 2.6-fold) observed in cells cotreated with TCDD plus E2. Immunoreactive p53 protein was not significantly affected by treatment with E2, TCDD, or TCDD plus E2. RB phosphorylation and modulation of cyclin D1 and p21 association with cdk2 and cdk4. E2 induced a time-dependent increase in RB phosphorylation and a 2.7-fold induction was observed after 24 h; in contrast, TCDD caused a time-dependent decrease in RB phosphorylation, which was decreased by 60% after 24 h (Fig. 5). In cells cotreated with TCDD plus E2, there was a time-dependent decrease in hormone-induced RB phosphorylation and significant inhibition was observed after 12 and 24 h. The effects of E2, TCDD, and TCDD plus E2 on association of cyclin D1 with p21 were investigated by analyzing immunoreactive cyclin D1 immunoprecipitated with p21 antibodies (Fig. 6A). E2 induced a 2.7-fold increase in cyclin D1 protein associated with p21 within 6 h after treatment, and after 24 h this was increased only by 1.8-fold. Similar results were recently reported by Planas-Silva and
Weinberg (15). TCDD alone had minimal effects on cyclin D1 associated with p21 after 24 h; however, there was a transient increase 6 h after treatment with TCDD. In cells cotreated with E2 plus TCDD, that temporal pattern of cyclin D1 associated with p21 (Fig. 6A) is similar to that observed for cyclin D1 alone (Fig. 2) in which levels are decreased compared to cells treated with E2 alone. The results in Fig. 6B were obtained by addition of p21 antibodies followed by SDS–PAGE analysis of the resulting supernatant for cdk2 and cdk4 immunoreactive proteins. This assay measures cdk2 and cdk4 proteins not associated with p21. E2 caused a time-dependent increased dissociation of cdk2 and cdk4 from p21 even though cellular levels of both proteins were not altered after hormone treatment; TCDD alone had no effect and, in combination with E2, TCDD slightly decreased dissociation of both proteins from p21.
FIG. 2. Western blot analysis of cyclin D1 protein levels on MCF-7 cells after treatment with E2, TCDD, or TCDD plus E2. (A) Western blot analysis of cyclin D1. The experimental design was the same as described in the legend to Fig. 1 and Western blot analysis was determined as described under Materials and Methods. The relative protein levels compared to control (U) levels (arbitrarily set at 1.0) in lanes 1 through 10 are 1.0, 2.8 6 0.7,a 3.0 6 0.9,a 2.8 6 0.7,a 2.6 6 0.7, 1.6 6 0.3,b 1.2 6 0.2,b 1.3 6 0.2, 0.8 6 0.2, and 0.5 6 0.3, respectively. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2-induced response. (B) Graphical representation of cyclin D1 protein levels obtained from results in A. All data are means 6 SE for three separate (replicate) determinations.
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mal effect on cyclin H protein levels, there was a timedependent increase in cdk7 levels, which were elevated 2.1-fold after 24 h (Fig. 7A). TCDD alone had no significant effect on either protein but, in combination with E2, significantly decreased hormone-induced cdk7 protein levels after 24 h. E2 caused a 3.6-fold increase of cdk7-dependent kinase activity after 12 h when histone H1 was used as substrate and this induced activity was decreased 24 h after treatment (Fig. 7B). TCDD alone caused an approximately 2-fold increase in cdk7-dependent kinase activity 6, 12, and 24 h after treatment; in cells cotreated with E2 plus TCDD, cdk7dependent kinase activity was significantly decreased only after treatment with TCDD for 12 h (Fig. 7B). Inhibition of tyrosine 15 phosphorylation in cdk2 and cdk4 results in activation of cdk2 and cdk4 kinase complexes, and therefore, effects of E2 and TCDD on tyrosine phosphorylation of cdk2 and cdk4 proteins were investigated. Using an anti-phosphotyrosine antibody (PY20) to immunodeplete the phosphorylatedtyrosine cdk2 and cdk4 proteins, the unphosphorylated-tyrosine cdk2 and cdk4 protein levels were then determined in the supernatants. The results show that E2 significantly increased forms of cdk2 and cdk4 protein in the supernatant which were not phosphorylated at tyrosine 15 (Fig. 8). TCDD alone had no effect on tyrosine phosphorylation of cdk2 and cdk4, and TCDD in combination with E2 did not significantly affect the FIG. 3. Effects of E2, TCDD, and TCDD plus E2 on cdk2- and cdk4-dependent kinase activities. (A) Kinase-dependent substrate phosphorylation. The whole-cell lysates (350 mg) from various treatment groups as described in the legend to Fig. 1 were used for each kinase assay. Immunoprecipitates were obtained using anti-cdk4 and anti-cdk2 antibodies. Cdk4 kinase activity was determined using GST-RB as a substrate. Cdk2 kinase activity was determined using histone (Sigma type III-SS) as a substrate. The detailed kinase reaction conditions are described under Materials and Methods. Kinase activities were analyzed by SDS–PAGE and band intensities were measured by densitometry as described under Materials and Methods. For cdk4-dependent kinase activity, the relative intensities of bands compared to control (U) (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 1.9 6 0.3,a 3.8 6 0.6,a 1.2 6 0.4, 0.8 6 0.1, 0.9 6 0.5, 0.7 6 0.2, 0.9 6 0.1,b 0.8 6 0.2b and 0.8 6 0.4, respectively. For cdk2-dependent kinase activity, the relative intensities of bands in lanes 1 through 10 were 1.0, 1.3 6 0.2, 1.7 6 0.2, 2.4 6 0.3,a 1.2 6 0.1, 1.3 6 0.2, 1.4 6 0.3,b 1.2 6 0.1, 0.9 6 0.1, and 0.9 6 0.1, respectively. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2-induced response. All data are means 6 SE for three separate (replicate) determinations. (B) Graphical representation of cdk4- and cdk2-dependent kinase activities obtained from results summarized in A.
Hormonal effects on cdk7, tyrosine phosphorylation, and cdc25A. The cyclin H/cdk7 protein or cdk-activating kinase (CAK) plays an important role in phosphorylation of both cdk2 and cdk4 at threonine-160 and threonine-172, respectively, to activate kinase activity. Although treatment of MCF-7 cells with E2 had mini-
FIG. 4. The effects of E2, TCDD, and TCDD plus E2 on p27, p21, and p53 protein levels. The whole-cell lysates (100 mg) as described in the legend to Fig. 1 were used for the assay and Western blot analyses were carried out as described under Materials and Methods. Relative p53 protein levels were not significantly changed by any treatment. Protein levels relative to controls (U) (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 1.4 6 0.2, 1.6 6 0.3, 1.6 6 0.2, 1.7 6 0.4, 2.0 6 0.5, 1.9 6 0.4, 1.4 6 0.2, 1.5 6 0.1, and 1.3 6 0.2, respectively, for p27 protein and 1.0, 1.2 6 0.2, 1.1 6 0.2, 1.1 6 0.1, 2.6 6 0.3,b 2.5 6 0.6,b 1.8 6 0.5,b 2.0 6 0.5,a 1.5 6 0.2, and 1.2 6 0.1, respectively, for p21 protein. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2-induced response. All data are means 6 SE for three separate (replicate) determinations.
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T47D human breast cancer cells and induction of several E2-responsive genes or proteins, including tissue plasminogen activator, pS2, prolactin receptor, cathepsin D, and progesterone receptor (26 –28, 46 –51). Many of the effects of ‘‘pure’’ antiestrogens ICI 164,384 and ICI 182,780, which act via complex modulation of ERmediated responses (52–55), are similar to those reported for AhR agonists in ER-positive breast cancer cells. The molecular mechanisms of cross talk between ER- and AhR-mediated responses are complex (26); however, at least one pathway has been defined for two genes, namely pS2 and cathepsin D. Inhibition of E2induced pS2 and cathepsin D gene expression by TCDD occurs through interaction of the nuclear AhR heterodimer with iDREs in 59-promoter regions of both genes (26, 27). In contrast, the more complex mecha-
FIG. 5. Effects of E2, TCDD, and TCDD plus E2 on phosphorylation of retinoblastoma protein. (A) SDS–PAGE analysis of pRB and ppRB. The whole-cell lysates (70 mg) from different treatment groups as described in the legend to Fig. 1 were analyzed on 7.5% SDS– PAGE as described under Materials and Methods. The hyperphosphorylated and hypophosphorylated forms of RB are represented as ppRB and pRB, respectively. Hypophosphorylated RB levels were not significantly altered by the various treatments, whereas hyperphosphorylated RB was significantly altered by E2, TCDD, and TCDD plus E2. Relative levels of ppRB compared to controls (U) (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 1.8 6 0.1,a 2.6 6 0.4,a 2.7 6 0.7,a 1.7 6 0.4, 1.5 6 0.3,b 1.1 6 0.3,b 1.1 6 0.2, 0.8 6 0.3, and 0.4 6 0.1, respectively. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2induced response. All data are means 6 SE for three separate (replicate) determinations. (B) Graphical representation of relative values of ppRB. Results are derived from data shown in A.
hormone-induced responses. Inhibition of tyrosine phosphorylation and the subsequent E2-mediated increases in cdk2/cdk4 forms which are not phosphorylated on tyrosine-15 may also be related to increased cdc25 phosphatase protein, which catalyzes hydrolysis of phosphorylated tyrosine residues. The results illustrated in Fig. 9 show that E2 caused 2.5-fold increase of cdc25A protein levels in MCF-7 cells after 12 h and this increase was observed 24 h after initial treatment. In contrast, TCDD alone had minimal effect on cdc25A protein and did not modulate E2-induced upregulation of this protein (Fig. 9). DISCUSSION
TCDD inhibits E2- and growth factor-induced proliferation and DNA synthesis in ER-positive MCF-7 and
FIG. 6. Effects of E2, TCDD, and TCDD plus E2 on association of p21 with cyclin D1, cdk2, and cdk4. The whole-cell lysates (350 mg) as described in the legend to Fig. 1 were immunoprecipitated by three sequential immunoprecipitations with p21 antibody as described under Materials and Methods. (A) p21– cyclin D1 association. The combined immunoprecipitates were used to investigate the association between p21 and cyclin D1 using Western blot analysis. Relative intensities of cyclin D1 protein bands compared to controls (U) (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 2.7 6 0.2,a 2.5 6 0.2,a 1.8 6 0.2,a 2.4 6 0.3, 2.2 6 0.2, 1.6 6 0.2, 1.7 6 0.2,a 1.1 6 0.1, and 0.8 6 0.1, respectively. a Significantly different (P , 0.05) from control values. (B) Dissociation of cdk2 and cdk4 from p21. The p21-immunodepleted supernatants were used to examine cdk2 and cdk4 protein levels remaining in the supernatants after p21 immunoprecipitation. Relative cdk2 protein levels in the supernatant compared to controls (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 1.5 6 0.1, 1.7 6 0.2,a 2.2 6 0.1,a 1.8 6 0.2, 1.8 6 0.3, 1.6 6 0.1, 1.3 6 0.1, 1.2 6 0.1, and 0.9 6 0.2, respectively. Relative cdk4 protein levels in the supernatant compared to controls (arbitrarily set at 1.0) were 1.0, 1.6 6 0.1,a 1.6 6 0.2,a 2.0 6 0.1,a 1.4 6 0.1, 1.3 6 0.1, 1.3 6 0.1, 1.1 6 0.1, 1.0 6 0.2, and 0.9 6 0.5, respectively. a Significantly different (P , 0.05) from control values. All data are given as means 6 SE for three separate (replicate) determinations.
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in part, to results of previous studies which reported increased cyclin D1 expression (protein and mRNA), increased cdk2- and cdk4-dependent kinase activities, and hyperphosphorylation of RB (13–15). Despite similarities between these studies, significant differences were also observed. Some of these differences may be due to cell
FIG. 7. Effects of E2, TCDD, and TCDD plus E2 on cdk-acting kinase (cyclin H/cdk7) protein levels and kinase activity. (A) Western blot analysis. The whole-cell lysates (100 mg) described in the legend to Fig. 1 were used for the assay. Cyclin H protein expression was not significantly changed by E2, TCDD, or TCDD plus E2 treatment. Relative protein levels of cdk7 in lanes 1 through 10 compared to controls (U) (arbitrarily set at 1.0) were 1.0, 1.4 6 0.1, 1.8 6 0.2,a 2.1 6 0.3,a 1.4 6 0.3, 1.5 6 0.2, 1.2 6 0.1,b 1.1 6 0.2, 1.1 6 0.2, and 0.8 6 0.1, respectively. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2-induced response. (B) Cdk7 kinase activity. Whole-cell lysates (200 mg) were incubated with 0.3 mg of anti-cdk7 antibody overnight. Fifteen microliters of protein A–agarose beads was added and incubated for an additional 3 h at 4°C. The kinase assay was carried out as described under Materials and Methods. The relative cdk7-dependent kinase activities in lanes 1 through 10 were 1.0, 1.4 6 0.1, 3.6 6 0.3,a 1.3 6 0.3, 1.4 6 0.2, 2.7 6 0.3,b 1.3 6 0.2, 2.1 6 0.1, 1.8 6 0.4, and 1.8 6 0.3, respectively. All data are means 6 SE for three separate (replicate) determinations. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2-induced response.
nisms of growth inhibition of tumors or breast cancer cells by AhR agonists are unknown (33–37, 41– 45) and were therefore investigated in this study. Modulation of cell cycle enzymes and their dependent activities by E2 in MCF-7 cells. Basal and mitogeninduced cell growth is regulated by multiple proteins which control cell cycle progression from G0/G1 to M phase and these include cyclins, cyclin-dependent kinases, and their inhibitors. Initial studies showed that in MCF-7 cells treated with 10 nM E2, there was a significant progression of the cells from G0/G1 to S phase, and this was inhibited by TCDD (Table I). Therefore, this research has focused on delineating critical E2-inducible cell cycle responses associated with G0/G1 3 S, which are inhibited via cross talk with the AhR. The effects of E2 alone observed in this study (Figs. 1–5) are comparable,
FIG. 8. Effects of E2, TCDD, and TCDD plus E2 on phosphotyrosine status of cdk2 and cdk4 proteins. (A) The whole-cell lysates (350 mg) as described in the legend to Fig. 1 were immunoprecipitated by three sequential immunoprecipitations with anti-phosphotyrosine (PY20) antibody as described under Materials and Methods. The PY20-immunodepleted supernatants were used to examine unphosphorylated-tyrosine cdk2 and cdk4 protein levels. Relative unphosphorylated-tyrosine cdk2 protein levels in the supernatant compared to controls (U) (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 1.5 6 0.3, 1.6 6 0.3, 1.8 6 0.2,a 1.5 6 0.3, 1.2 6 0.2, 1.1 6 0.2, 1.0 6 0.0, 0.9 6 0.1, and 0.7 6 0.1, respectively. Unphosphorylated-tyrosine cdk4 protein levels in the supernatant compared to controls (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 1.8 6 0.3, 1.8 6 0.2, 2.2 6 0.2,b 1.8 6 0.1, 1.7 6 0.2, 1.8 6 0.3, 1.3 6 0.5, 1.1 6 0.1, and 1.0 6 0.3, respectively. All data were means 6 SE for three separate determinations. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2induced response. (B) Graphical representation of dephosphorylatedtyrosine cdk2 and cdk4 protein levels. Results are derived from data shown in A.
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FIG. 9. Western blot analysis of cdc25A protein levels. (A) Western blot analysis of cdc25A. Whole-cell lysates (20 mg) as described in the legend to Fig. 1 were used for the assay and Western blot analysis was carried out as described under Materials and Methods. The relative protein levels compared to controls (U) (arbitrarily set at 1.0) in lanes 1 to 10 are 1.0, 1.9 6 0.4, 2.5 6 0.2,a 2.5 6 0.3,a 2.2 6 0.4, 2.5 6 0.1, 2.6 6 0.2, 1.7 6 0.5, 1.4 6 0.3, and 1.2 6 0.0, respectively. All data are represented as means 6 SE for three determinations. a Significantly different (P , 0.05) from control values. (B) Graphical representation of cdc25A protein levels obtained from A.
maintenance prior to addition of E2. In one study (14), cells were maintained in 10 nM ICI 182,780 (a potent antimitogen) for 48 h prior to addition of 100 nM E2, whereas cells in this study were maintained in serumfree medium before adding 10 nM E2. Prall and co-workers (14) also reported that treatment of MCF-7 cells with E2 did not alter levels of immunoreactive p21 or p27 protein; however, active cyclin E–cdk2 complexes were deficient in both p21 and p27 protein complexes, and cdk2–threonine-160 phosphorylation was increased. Planas-Silva and Weinberg (15) also observed no significant changes in p21 or p27 levels in MCF-7 cells treated with E2, whereas Foster and Wimalasena (13) reported that E2 decreased p27 but not p21 protein levels in MCF-7 cells. Results of this study also show that E2 did not significantly affect immunoreactive p53, p21, or p27 levels (Fig. 4). Planas-Silva and Weinberg (15) recently showed that after treatment of tamoxifen-arrested MCF-7 cells with E2, there was rapid activation (within 6 h) of cyclin E– cdk2 complexes which was associated with redistribution of p21 from cyclin E– cdk2 to cyclin D1– cdk4 complexes. Our results showed that
treatment of MCF-7 cells with E2 followed by immunoprecipitation with p21 antibodies resulted in a timedependent increase in association of cyclin D1 with p21 (Fig. 6A), which was also observed by Planas-Silva and Weinberg (15). In contrast to their results, p21 immunodepletion studies showed that after treatment with E2, there was a significant increase of cdk2 and cdk4 proteins in the supernatant and thereby dissociation from the p21 protein complex (Fig. 6B). Thus, increased cdk4-dependent kinase activity may be due to increased cyclin D1 protein (Fig. 2) and dissociation of cdk4 from p21 (Fig. 6B), whereas E2-induced cdk2dependent kinase activity is related to increased dissociation from p21 (Fig. 6B). Another possible explanation for the coordinate increase in both cdk2- and cdk4dependent activities (Fig. 3) may be due to increased cdk7 protein and cdk7-dependent kinase activity, which activates both cdk2 and cdk4 by phosphorylation of critical threonine residues (56, 57). Results of this study show that E2 significantly induced phosphorylation of cdk2 (*cdk2, an active form of cdk2) (Fig. 1), which parallels activation of cdk7-dependent kinase activity after treatment with E2 (Fig. 7). Thus, E2induced activation of cdk7 (Fig. 7) followed by downstream activation of cdk2- and cdk4-dependent kinase activities may also be an important factor associated with the mitogenic activity of E2 in MCF-7 human breast cancer cells. Moreover, activation of cdk– cyclin complexes is dependent not only on phosphorylation of threonine-160 in cdk2 and threonine-172 in cdk4, but also on dephosphorylation of threonine-14 and tyrosine-15 in cdk2 and cdk4 (58, 59). Results of this study show that E2 increases levels of cdc25A phosphatase protein and this parallels increased levels of unphosphorylated-tyrosine cdk2 and cdk4 protein which are observed after treatment of MCF-7 with E2 (Figs. 8 and 9). These results suggest that cdc25A is another factor involved in activation of cdk2 and cdk4 kinase complexes by E2. In contrast, Planas-Silva and Weinberg (15) reported that E2 did not affect CAK activity and inactivation of cdk2 was not related to cdc25A in tamoxifen-arrested MCF-7 cells. Conflicting reports on the effects of E2 on cell cycle proteins and their activities observed in this and other studies may be due to the known interlaboratory genotypic variations of wild-type and truncated ER transcripts and altered E2 responsiveness of MCF-7 cells (60, 61). Another source of variability may be associated with different conditions for growth-arresting MCF-7 cells, which include maintenance in serum-free medium (this study and (13)) and treatment with ICI 182,780 (14) or tamoxifen (15). Effects of TCDD alone on cell cycle proteins and enzyme activities. Treatment of MCF-7 cells with TCDD alone results in minimal inhibition of cell growth (33–
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38) and changes in cell cycle distribution (Table I). Moreover, only cyclin D1 protein levels and RB phosphorylation were significantly decreased and p21 was transiently (6 h) increased after treatment of MCF-7 cells with TCDD alone. In contrast, other antiproliferative agents such as retinoids or the antiestrogens ICI 164,384/182,780 alone inhibited breast cancer cell growth and differentially modulated multiple cell cycle enzymes/proteins. For example, treatment of MCF-7 cells with ICI 182,780 decreased cdk2- and cdk4-dependent kinase activities and cyclin D1 (mRNA and protein) and increased the cdk inhibitors p27 and p21 and the proportion of hypophosphorylated RB protein (24). In T47D cells, retinoids also increased the hypophosphorylated form of RB, and changes in other cell cycle proteins were observed only after prolonged treatment, compared to the more rapid responses observed for ICI 182,780 (23). Thus, TCDD, ICI 182,780, and retinoid acid elicit common and divergent effects on cell cycle enzymes, and this is consistent with their different mechanisms of action. Antiestrogenic activity of TCDD: Inhibition of multiple E2-regulated cell cycle proteins/enzymes. It has previously been reported that TCDD inhibited E2-induced proliferation and DNA synthesis in MCF-7 and T47D cells (33–38), and the data in Table I also show that TCDD blocks the increase of cells in S phase after treatment with E2. Our results also show that cotreatment of MCF-7 cells with TCDD plus E2 resulted in significant inhibition of E2-induced hyperphosphorylation of RB and cyclin D1 proteins and cdk2-, cdk4-, and cdk7-dependent kinase activities (Figs. 2, 3, 5, and 6). Inhibition of E2-induced cdk4-dependent kinase activity by TCDD may be related to the parallel decrease of E2-induced cyclin D1 which forms the cyclin D1– cdk4 kinase complex. Inhibition of E2-induced cdk2- and cdk4-dependent kinase activities by TCDD also correlated with the significantly increased p21 levels in cotreated cells and increased association of cdk2 and cdk4 proteins with p21 as determined in the p21 antibody immunoprecipitation studies (Fig. 6). In summary, our results demonstrate that activation of cdk2- and cdk4-dependent kinase activities in MCF-7 cells after treatment with E2 is a complex process which involves multiple cell cycle proteins as depicted in Fig. 10. For example, E2 induced cyclin D1, decreased association of both cdk2 and cdk4 proteins with p21, increased cdk7-dependent kinase activity, and decreased tyrosine phosphorylation of cdk2 and cdk4 proteins, and this was accompanied by increased cdc25A phosphatase protein levels. All of these responses were accompanied by an increase in percentage of cells in S phase and a decreased percentage in G0/G1 (Table I). In contrast, TCDD reversed the effects of E2 on distribution of MCF-7 cells in the cell cycle and
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FIG. 10. Modulation of the cell cycle by E2 and TCDD plus E2. Specific steps that are induced by E2 are denoted E1. E2-induced responses inhibited by TCDD are denoted T2 and induction by TCDD alone is indicated by T1.
this was accompanied by inhibition of several other responses which were elevated after treatment with E2. Current studies are focused on further delineating the molecular mechanisms of AhR-mediated antiestrogenicity associated with inhibition of E2-responsive cell cycle proteins/enzymes and on developing AhRbased drugs for clinical treatment of breast cancer. ACKNOWLEDGMENTS S. Safe is a Sid Kyle Professor of Toxicology. We thank Betty Rosenbaum for her technical assistance.
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38. Vogel, C., and Abel, J. (1995) Arch. Toxicol. 69, 259 –265. 39. Chaloupka, K., Krishnan, V., and Safe, S. (1992) Carcinogenesis 13, 2223–2239. 40. Liu, H., Wormke, M., Safe, S., and Bjeldanes, L. F. (1994) J. Natl. Cancer Inst. 86, 1758 –1765. 41. Kociba, R. J., Keyes, D. G., Beger, J. E., Carreon, R. M., Wade, C. E., Dittenber, D. A., Kalnins, R. P., Frauson, L. E., Park, C. L., Barnard, S. D., Hummel, R. A., and Humiston, C. G. (1978) Toxicol. Appl. Pharmacol. 46, 279 –303. 42. Gierthy, J. F., Bennett, J. A., Bradley, L. M., and Cutler, D. S. (1993) Cancer Res. 53, 3149 –3153. 43. Holcomb, M., and Safe, S. (1994) Cancer Lett. 82, 43– 47. 44. Lucier, G. W., Tritscher, A., Goldsworthy, T., Foley, J., Clark, G., Goldstein, J. A., and Maronpot, R. (1991) Cancer Res. 51, 1391– 1397. 45. McDougal, A., Wilson, C., and Safe, S. (1997) Cancer Lett. 120, 53– 63. 46. Gierthy, J. F., Lincoln, D. W., Gillespie, M. B., Seeger, J. I., Martinez, H. L., Dickerman, H. W., and Kumar, S. A. (1987) Cancer Res. 47, 6198 – 6203. 47. Zacharewski, T. R., Bondy, K. L., McDonell, P., and Wu, Z. F. (1994) Cancer Res. 54, 2707–2713. 48. Lu, Y.-F., Sun, G., Wang, X., and Safe, S. (1996) Arch. Biochem. Biophys. 332, 35– 40. 49. Wang, X., Porter, W., Krishnan, V., Narasimhan, T. R., and Safe, S. (1993) Mol. Cell. Endocrinol. 96, 159 –166. 50. Harper, N., Wang, X., Liu, H., and Safe, S. (1994) Mol. Cell. Endocrinol. 104, 47–55. 51. Krishnan, V., Narasimhan, T. R., and Safe, S. (1992) Anal. Biochem. 204, 137–142. 52. Dauvois, S., Danielian, P. S., White, R., and Parker, M. G. (1992) Proc. Natl. Acad. Sci. USA 89, 4037– 4041. 53. Katzenellenbogen, B. S., Montano, M. M., Le Goff, P., Schodin, D. J., Kraus, W. L., Bhardwaj, B., and Fujimoto, N. (1995) J. Steroid Biochem. Mol. Biol. 53, 387–393. 54. McDonnell, D. P., Clemm, D. L., Hermann, T., Goldman, M. E., and Pike, J. W. (1995) Mol. Endocrinol. 9, 659 – 669. 55. Wakeling, A. E. (1995) Biochem. Pharmacol. 49, 1545–1549. 56. Fisher, R. P., and Morgan, D. O. (1994) Cell 78, 713–724. 57. Makela, T. P., Tassan, J. P., Nigg, E. A., Frutiger, S., Hughes, G. J., and Weinberg, R. A. (1994) Nature 371, 254 –257. 58. Gu, Y., Rosenblatt, J., and Morgan, D. O. (1992) EMBO J. 11, 3995– 4005. 59. Galaktionov, K., Lee, A. K., Eckstein, J., Draetta, G., Meckler, J., Loda, M., and Beach, D. (1995) Science 269, 1575–1577. 60. Villalobos, M., Olea, N., Brotons, J. A., Oleaserrano, M. F., Dealmodovar, J. M. R., and Pedraza, V. (1995) Environ. Health Perspect. 103, 844 – 850. 61. Klotz, D. M., Castles, C. G., Spriggs, L. L., and Hill, S. M. (1995) Biochem. Biophys. Res. Commun. 210, 609 – 615.
Vol. 356, No. 2, August 15, pp. 239 –248, 1998 Article No. BB980782
Aryl Hydrocarbon Receptor-Mediated Antiestrogenicity in MCF-7 Cells: Modulation of Hormone-Induced Cell Cycle Enzymes1 Weili Wang, Roger Smith, III, and Stephen Safe2 Department of Veterinary Physiology and Pharmacology and Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas 77843-4466
Received April 20, 1998, and in revised form May 23, 1998
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits 17b-estradiol (E2) mammary tumor growth in rodents and in MCF-7 human breast cancer cells; however, the cell cycle genes/proteins which are inhibited have not been determined. Initial studies showed that treatment of MCF-7 cells with 10 nM E2 significantly increased cyclin D1 (protein and mRNA), cdk2- and cdk4-dependent kinase activities, and hyperphosphorylation of retinoblastoma (RB) protein. In contrast to results of recent studies (M. D. Planas-Silva and R. A. Weinberg, 1997, Mol. Cell. Biol. 17, 4059 – 4069), E2 induced dissociation of both cdk2 and cdk4 proteins from the p21 protein complex and significantly increased cdk7-dependent kinase activity. Treatment of MCF-7 cells with E2 also induced cdc25A phosphatase protein, which was accompanied by increased cdk2 and cdk4 proteins containing unphosphorylated tyrosine residues. Although TCDD alone has minimal effects on cell cycle proteins/enzymes, several E2-induced responses were significantly inhibited in MCF-7 cells cotreated with E2 plus TCDD. For example, TCDD significantly inhibited E2-induced hyperphosphorylation of RB, cyclin D1 protein, and cdk2-, cdk4-, and cdk7-dependent kinase activities. Inhibition of E2-induced cdk4-dependent kinase activity by TCDD may be related to the parallel decrease of E2-induced cyclin D1 protein, and inhibition of induced cdk2- and cdk4-dependent kinase activities may be due to significantly increased p21 levels in cells cotreated with TCDD plus E2. These results demonstrate that the antiestrogenic activity of TCDD is due to downregula-
1 The financial assistance of the National Institutes of Health (CA-64081 and ES04176) and the Texas Agricultural Experiment Station is gratefully acknowledged. 2 To whom correspondence should be addressed. Fax: (409) 8624929. E-mail: [email protected].
0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
tion of several E2-induced cell cycle proteins/activities and this illustrates the complex cross talk between the aryl hydrocarbon and the E2 receptor signaling pathways. © 1998 Academic Press Key Words: estrogen; 2,3,7,8-tetrachlorodibenzo-pdioxin; cell cycle; cross-talk mechanisms.
Estrogenic hormones play an essential role in mammalian physiology (1); however, it has also been demonstrated that lifetime exposure to estrogens is a major risk factor for both breast and endometrial cancer in women (2– 4). Although the precise roles of 17b-estradiol (E2)3 and related estrogenic hormones in development of mammary cancer have not been established (5, 6), it is evident from both laboratory animal and in vitro cell culture studies that estrogen receptor (ER)mediated gene expression and growth factors are important components of the cancer process (7–12). Several studies have investigated the effects of E2, antiestrogens, and other inhibitors of mammary cancer cell growth and cell cycle enzymes in ER-positive MCF-7 or T47D human breast cancer cell lines (13–25). Foster and Wimalasena (13) reported that in growtharrested MCF-7 cells, 89.4% of the cells were in G0/G1, and only 6.6 and 4.0% were in S and G2/M phase, respectively. The antiestrogen ICI 182,780 maintained this distribution of cells in different phases of the cell cycle, whereas treatment with E2 decreased the per3 Abbreviations used: E2, 17b-estradiol; ER, estrogen receptor; RB, retinoblastoma; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AhR, aryl hydrocarbon receptor; DRE, dioxin-responsive element; i, inhibitory; DME, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; DCC, dicyclohexylcarbodiimide; FBS, fetal bovine serum; CAK, cdk-activating kinase; GST, glutathione S-transferase; ECL, enhanced chemiluminescence.
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centage of cells in G0/G1 (65.2%) and increased cells in S (20.6%) and G2/M (14.2%) phases. These changes in cell cycle distribution were accompanied by increased cdk2- and cdk4-associated kinase activities, increased phosphorylation of retinoblastoma (RB) protein, and increased cyclin D1 protein levels. E2 also decreased cdk-inhibitory activities associated with p27kip-1 and this was accompanied by lower p27kip-1 protein levels. Similar results were reported by Prall and co-workers (14), who utilized MCF-7 cells growth arrested by the antiestrogen ICI 182,780. Their results also showed that hormone-induced activation of cyclin E– cdk2 complexes resulted in decreased association with cdk inhibitors p27kip-1 and p21 and increased phosphorylation of cdk2 threonine-160. In tamoxifen growth-arrested cells, Planas-Silva and Weinberg (15) reported that treatment with E2 also resulted in redistribution of cdk inhibitor p21 from its association with cyclin E– cdk2 to cyclin D– cdk4. These studies exhibited some similarities and differences which may be due to differences in experimental methodologies and methods for arresting growth of MCF-7 cells. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a compound which binds with high affinity to the aryl hydrocarbon receptor (AhR), and several studies show that TCDD and related AhR agonists inhibit diverse E2-induced responses (reviewed in 26). The mechanisms of cross talk between AhR and ER signaling pathways have been investigated using cathepsin D and pS2 genes as models (27, 28). Inhibition of E2induced pS2 and cathepsin D gene expression by TCDD involves interaction of the nuclear AhR complex with GCGTG sequences (27, 28) containing the core binding motif of the dioxin-responsive element (DRE) which forms part of the genomic enhancer element required for AhR-mediated transactivation (29 –32). The inhibitory DRE (iDRE) core binding motifs in the pS2 and cathepsin D gene promoters interact with the nuclear AhR complex; however, enhanced gene expression is not observed, due to the absence of specific nucleotides which flank the core iDRE. AhR agonists also inhibit E2- and growth factor-induced proliferation of MCF-7 cells (33– 40) and formation and growth of mammary tumors in rodent models (41– 45). This study utilized TCDD as a probe for determining AhRmediated effects on key cell cycle proteins which are involved in growth-promoting activity of estrogens. In this study, E2 induced cyclin D1 protein, cdk2- and cdk4-dependent kinase activities, and RB phosphorylation in MCF-7 cells, and all of these responses were inhibited by TCDD. These interactive effects were also accompanied by rapid upregulation of p21 protein in MCF-7 cells treated with TCDD alone or with TCDD plus E2; induction of this cdk inhibitor, coupled with inhibition of E2-induced cell cycle enzyme activities, demonstrates that the antiestrogenic activity of AhR
agonists is associated with modulation of multiple cell cycle enzymes and proteins. MATERIALS AND METHODS Chemicals and antibodies. E2 and histone (type III-SS) were purchased from Sigma Co. (St. Louis, MO). TCDD was prepared in this laboratory and was .98% pure as determined by high-pressure liquid and gas chromatography. Cyclin D1, cyclin A, cyclin E, cyclin H, cdk2, cdk4, cdk7, p21, p27, p53, cdc 25A, phosphotyrosine (PY20) and RB antibodies, protein A–agarose beads, and GST-RB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). [g-32P]ATP (3000 Ci/mmol) was obtained from New England Nuclear (Boston, MA). All other chemicals and biochemicals were purchased from commercial sources and were either reagent or molecular grade. Cell culture maintenance and growth. MCF-7 human breast cancer cells were obtained from the American Type Culture Collection (ATCC) and maintained in DME/F12 medium without phenol red and supplemented with 5% fetal bovine serum plus 1% antibiotic/ antimycotic solution at 37°C. For experiments, cells were seeded into 100-mm petri dishes and grown until 70% confluence was reached. Cells were then synchronized in DME/F12 serum-free medium for 3 days; cells were then treated with DMSO (0.1% total volume), 10 nM E2, E2 plus 10 nM TCDD, and 10 nM TCDD alone for 6, 12, and 24 h, respectively. Cells were then harvested and different cell parameters were determined as described below. Preparation of whole-cell extracts. Cells, treated as described above, were washed once in ice-cold PBS and scraped into lysis buffer (50 nM Hepes (pH 7.5), 150 mM sodium chloride, 10% (v/v) glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 200 mM sodium orthovanadate, 10 mM pyrophosphate, and 100 mM NaF). Cells were incubated for 30 min and then centrifuged at 10,000g for 5 min. Supernatants were precleared by addition of 20 ml of protein A–agarose beads for 30 min followed by centrifugation for 5 min at 10,000g. The lysates used for both Western blot and kinase assays were stored at 280°C until required. All procedures were carried out at 4°C. Western immunoblot analysis and immunoprecipitation. Equal amounts of protein from cell lysates prepared as described above were separated by SDS–PAGE and then transferred to nitrocellulose membrane using an electroblotting apparatus overnight at 4°C. Membranes were blocked with TBS (10 mM Tris–HCl, pH 8.0; 150 mM sodium chloride) plus 5% milk (Blotto buffer) for 1 h and then incubated in primary antibody at 0.1 to 0.5 mg/ml in the Blotto buffer for 1 to 2 h at 20°C. Membranes were rinsed once and washed for 6 min (33) in TBS buffer. The secondary anti-mouse or anti-rabbit/ HRP (1:1000 –5000) was added to Blotto buffer and incubated for 1 h at 20°C. Membranes were washed as described above and incubated in NEN ECL reagents for 1 min; excess ECL reagent was removed by dabbing with a Kimwipe, and membranes were sealed in plastic wrap. Membranes were then exposed to ECL hyperfilm for visualization of immunoreactive bands. Protein levels were quantitated using a Sharp JX-330 densitometer and a Scanalytics Zero-D software package (Scanalytics, Billerica, MA). For p21 immunoprecipitation and immunodepletion, cell lysates (350 mg protein in 150 ml) were immunoprecipitated by three sequential immunoprecipitations with 0.3 mg p21 antibody and 9 ml of protein A–agarose beads. Beads were then combined, washed three times with lysis buffer, boiled for 5 min in 23 SDS sample buffer, loaded on SDS–PAGE, and then assayed by Western blot analysis for cyclin D1 to investigate the association between p21 and cyclin D1. The p21-immunodepleted supernatants were saved and equal volumes (30 ml) of supernatants were loaded onto 10% SDS–PAGE and followed by Western blot analysis for cdk2 and cdk4 to examine the association of p21 with
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ESTROGEN AND 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN IN THE CELL CYCLE cdk2 and cdk4. The procedure used for PY20 immunodepletion was similar to that described for p21; PY20-immunodepleted supernatants were used to quantitate levels of unphosphorylated-tyrosine cdk2 and cdk4 by Western blot analysis. Quantitation of immunoreactive proteins was determined by densitometry. Kinase assays. Cell lysates (350 mg for cdk2 and cdk4 kinase assays; 200 mg for cdk7 kinase assay), prepared as described above, were incubated with individual antibodies (300 to 500 ng/each) for 3 to 15 h at 4°C and then immunoprecipitated by incubation with protein A–agarose beads for 3 h at 4°C. Beads were washed with lysis buffer (33) and with kinase buffer (33) (50 mM Tris–Hepes, pH 7.5, 10 mM MgCl2). Kinase reaction was carried out in 30 ml of kinase buffer supplemented with 400 mg/ml histones (Sigma type III-SS) for cdk2 and cdk7 kinase assays or 0.3 mg GST-RB (sc-4112; Santa Cruz Biotechnology) for cdk4 kinase assay (10 mM ATP, 0.5 mM dithiothreitol, 0.5 mM EGTA, and 5 mCi [g-32P]ATP for 20 to 40 min at 30°C). Assays were stopped with 35 ml of 23 SDS–PAGE sample buffer and boiled for 5 min; samples were then loaded and separated on 10% SDS–PAGE. The 30-kDa phosphorylated histone and 50-kDa phosphorylated GST-RB products were readily observed and were visualized by autoradiography using Kodak film. Intensities of specific bands were quantitated by densitometry as described above. Flow cytometry analysis. Cells were seeded in six-well plates in DME/F12 medium supplemented with 2.5% DCC–FBS. Cells were synchronized in serum-free DME/F12 medium for 3 days. Cells were then treated with 10 nM E2, 10 nM E2 plus 10 nM TCDD, and 10 nM TCDD in serum-free medium for 12 and 24 h, respectively. Cells were washed once with PBS, harvested, resuspended in 0.5 ml of PBS, fixed by dropwise addition of 4.5 ml of ice-cold 70% ethanol, and stored overnight at 220°C. Fixed cells were then washed once and resuspended in a propidium iodide staining buffer, consisting of 0.1% (v/v) Triton X-100 (Sigma), 200 mg/ml DNase-free RNase A (Sigma), and 50 mg/ml propidium iodide (Sigma). Cells were incubated for 1 to 6 h at 20°C prior to analysis on a FACS Calibur instrument (Becton– Dickinson, San Jose, CA). Data were analyzed in ModFit LT for the Macintosh, version 2.0 (Verity Software House, Topsham, ME), using the diploid model, with visible G2M peak, for fresh/frozen cells. The coefficient of variation varied from 3.8 to 5.3% for all samples. Statistics. Results are expressed as means 6 SE for at least three independent (replicate) experiments for each treatment group. Statistical significance was determined by ANOVA and Student’s t test and the levels of probability are noted.
RESULTS
Effects of E2, TCDD, and TCDD plus E2 on cell cycle distribution of MCF-7 cells and cell cycle proteins. MCF-7 cells were maintained in DME/F12 medium containing 5% fetal bovine serum and cells were then cultured for 3 days in serum-free medium prior to treatment with E2, TCDD, or TCDD plus E2 for 6, 12, or 24 h. These experiments were all carried out in serum-free medium. Using this protocol, the maximal effects on cell cycle parameters were observed for 10 nM E2, and 10 nM TCDD was the optimal concentration for inhibiting E2-induced responses. The cell cycle distribution of MCF-7 cells was determined by flow cytometry; cells in S phase were not increased after treatment with E2 for 12 h; however, a significant increase in the percentage of cells in S phase and a decreased percentage in G0G1 were observed after treatment for 24 h (Table I). TCDD alone had minimal effects; however, in cells cotreated with TCDD plus E2,
TABLE I
Effects of 17b-Estradiol and TCDD on Cell Cycle Distribution of MCF-7 Human Breast Cancer Cellsa Cell cycle phase (%) Treatment (time, h)
G0/G1
S
G2/M
Control E2 (12) E2 1 TCDD (12) TCDD (12) E2 (24) E2 1 TCDD (24) TCDD (24)
89.9 6 2.1 87.7 6 2.1 87.2 6 0.2 89.1 6 0.8 75.1 6 0.6b 81.0 6 1.3c 90.8 6 0.6
4.9 6 1.6 6.0 6 1.4 7.9 6 0.7 6.7 6 0.8 23.4 6 1.7b 15.8 6 1.8d 5.2 6 0.5
5.2 6 0.6 4.4 6 0.7 4.9 6 0.5 4.2 6 0.2 1.5 6 1.2 3.2 6 0.7 4.0 6 0.9
a Cells were seeded in six-well plates at 3 3 105 cells/well in DME/F12 medium supplemented with 2.5% DCC–FBS. Cells were synchronized in serum-free DME/F12 medium (SFM) for 3 days. Cells were then treated with 10 nM E2, 10 nM E2 plus 10 nM TCDD, and 10 nM TCDD in SFM for 12 and 24 h, respectively. Cells were washed once and the assay was carried out as described under Materials and Methods. The cell cycle phase distribution in DMSOtreated (control) cells was determined after 24 h. Experiments were performed in triplicate and results are expressed as means 6 SE. b Significantly different (P , 0.001) from control cells at respective time points. c Significantly different (P , 0.04) from E2 24-h-treated cells at G0/G1 phase. d Significantly different (P , 0.001) from E2 24-h-treated cells at S phase.
there was significant inhibition of the hormone-induced increase in cells in S phase. The results summarized in Fig. 1 show that immunoreactive levels of cyclin E, cyclin A, cdk2, and cdk4 protein were not significantly changed in any of the treatment groups. E2 significantly induced phosphorylation of the active form of cdk2 (*cdk2, Fig. 1) as previously reported (14, 15). Cyclin D1 levels were decreased slightly in MCF-7 cells treated with TCDD alone, whereas E2 alone caused a rapid (within 6 h) and sustained 2.8- to 3-fold increase in cyclin D1 protein (Fig. 2). In cells cotreated with E2 plus TCDD, the hormone-induced response was significantly decreased after 12 or 24 h treatment with TCDD. The effects observed for E2 alone on this same set of cell cycle proteins were similar to those previously described (13), and suppression of E2-induced cyclin D1 by TCDD was comparable to the effects observed for the pure antiestrogen ICI 182,780 (13). Modulation of cdk4- and cdk2-dependent kinases, p21, p27, and p53. E2 alone caused a relatively rapid (within 6 h) 3.8-fold increase in cdk4-dependent kinase activity, which decreased to background levels after 24 h (Fig. 3). In contrast, cdk2-dependent kinase activity was induced more slowly and, after 24 h, a 2.4-fold increase was observed. TCDD alone did not affect cdk2or cdk4-dependent kinase activity; however, in cells cotreated with E2 plus TCDD, the hormone-induced activities were decreased at all time points. Treatment
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FIG. 1. Western blot analysis of effects of E2 and TCDD on cyclin E, cyclin A, cdk2, and cdk4 protein expression in MCF-7 cells. Cells were seeded into 100-mm petri dishes and synchronized for 3 days in serum-free DME/F12 medium. Cells were then treated with DMSO (0.1% v/v) as control (U), 10 nM E2, 10 nM E2 plus 10 nM TCDD, and 10 nM TCDD alone for 6, 12, and 24 h, respectively. Whole-cell lysates (100 mg), prepared as described under Materials and Methods, were used for the assay. The protein levels in the treated groups were not significantly changed compared to DMSO– control cells. However, the hyperphosphorylated form of cdk2 was significantly increased 2.4 6 0.3-fold (P , 0.05) with E2 treatment at 24 h compared to the control. TCDD only slightly blocked E2 effects at this time and not significantly. The Western blot assay was carried out as described under Materials and Methods.
of MCF-7 cells with E2 or TCDD had minimal effects on p27 cdk inhibitor protein levels; however, p27 protein levels were significantly elevated in cells cotreated with E2 1 TCDD (Fig. 4). E2 alone did not affect levels of p21 protein, whereas TCDD alone and in combination with E2 significantly elevated p21 levels with the highest increase (1.8- to 2.6-fold) observed in cells cotreated with TCDD plus E2. Immunoreactive p53 protein was not significantly affected by treatment with E2, TCDD, or TCDD plus E2. RB phosphorylation and modulation of cyclin D1 and p21 association with cdk2 and cdk4. E2 induced a time-dependent increase in RB phosphorylation and a 2.7-fold induction was observed after 24 h; in contrast, TCDD caused a time-dependent decrease in RB phosphorylation, which was decreased by 60% after 24 h (Fig. 5). In cells cotreated with TCDD plus E2, there was a time-dependent decrease in hormone-induced RB phosphorylation and significant inhibition was observed after 12 and 24 h. The effects of E2, TCDD, and TCDD plus E2 on association of cyclin D1 with p21 were investigated by analyzing immunoreactive cyclin D1 immunoprecipitated with p21 antibodies (Fig. 6A). E2 induced a 2.7-fold increase in cyclin D1 protein associated with p21 within 6 h after treatment, and after 24 h this was increased only by 1.8-fold. Similar results were recently reported by Planas-Silva and
Weinberg (15). TCDD alone had minimal effects on cyclin D1 associated with p21 after 24 h; however, there was a transient increase 6 h after treatment with TCDD. In cells cotreated with E2 plus TCDD, that temporal pattern of cyclin D1 associated with p21 (Fig. 6A) is similar to that observed for cyclin D1 alone (Fig. 2) in which levels are decreased compared to cells treated with E2 alone. The results in Fig. 6B were obtained by addition of p21 antibodies followed by SDS–PAGE analysis of the resulting supernatant for cdk2 and cdk4 immunoreactive proteins. This assay measures cdk2 and cdk4 proteins not associated with p21. E2 caused a time-dependent increased dissociation of cdk2 and cdk4 from p21 even though cellular levels of both proteins were not altered after hormone treatment; TCDD alone had no effect and, in combination with E2, TCDD slightly decreased dissociation of both proteins from p21.
FIG. 2. Western blot analysis of cyclin D1 protein levels on MCF-7 cells after treatment with E2, TCDD, or TCDD plus E2. (A) Western blot analysis of cyclin D1. The experimental design was the same as described in the legend to Fig. 1 and Western blot analysis was determined as described under Materials and Methods. The relative protein levels compared to control (U) levels (arbitrarily set at 1.0) in lanes 1 through 10 are 1.0, 2.8 6 0.7,a 3.0 6 0.9,a 2.8 6 0.7,a 2.6 6 0.7, 1.6 6 0.3,b 1.2 6 0.2,b 1.3 6 0.2, 0.8 6 0.2, and 0.5 6 0.3, respectively. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2-induced response. (B) Graphical representation of cyclin D1 protein levels obtained from results in A. All data are means 6 SE for three separate (replicate) determinations.
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mal effect on cyclin H protein levels, there was a timedependent increase in cdk7 levels, which were elevated 2.1-fold after 24 h (Fig. 7A). TCDD alone had no significant effect on either protein but, in combination with E2, significantly decreased hormone-induced cdk7 protein levels after 24 h. E2 caused a 3.6-fold increase of cdk7-dependent kinase activity after 12 h when histone H1 was used as substrate and this induced activity was decreased 24 h after treatment (Fig. 7B). TCDD alone caused an approximately 2-fold increase in cdk7-dependent kinase activity 6, 12, and 24 h after treatment; in cells cotreated with E2 plus TCDD, cdk7dependent kinase activity was significantly decreased only after treatment with TCDD for 12 h (Fig. 7B). Inhibition of tyrosine 15 phosphorylation in cdk2 and cdk4 results in activation of cdk2 and cdk4 kinase complexes, and therefore, effects of E2 and TCDD on tyrosine phosphorylation of cdk2 and cdk4 proteins were investigated. Using an anti-phosphotyrosine antibody (PY20) to immunodeplete the phosphorylatedtyrosine cdk2 and cdk4 proteins, the unphosphorylated-tyrosine cdk2 and cdk4 protein levels were then determined in the supernatants. The results show that E2 significantly increased forms of cdk2 and cdk4 protein in the supernatant which were not phosphorylated at tyrosine 15 (Fig. 8). TCDD alone had no effect on tyrosine phosphorylation of cdk2 and cdk4, and TCDD in combination with E2 did not significantly affect the FIG. 3. Effects of E2, TCDD, and TCDD plus E2 on cdk2- and cdk4-dependent kinase activities. (A) Kinase-dependent substrate phosphorylation. The whole-cell lysates (350 mg) from various treatment groups as described in the legend to Fig. 1 were used for each kinase assay. Immunoprecipitates were obtained using anti-cdk4 and anti-cdk2 antibodies. Cdk4 kinase activity was determined using GST-RB as a substrate. Cdk2 kinase activity was determined using histone (Sigma type III-SS) as a substrate. The detailed kinase reaction conditions are described under Materials and Methods. Kinase activities were analyzed by SDS–PAGE and band intensities were measured by densitometry as described under Materials and Methods. For cdk4-dependent kinase activity, the relative intensities of bands compared to control (U) (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 1.9 6 0.3,a 3.8 6 0.6,a 1.2 6 0.4, 0.8 6 0.1, 0.9 6 0.5, 0.7 6 0.2, 0.9 6 0.1,b 0.8 6 0.2b and 0.8 6 0.4, respectively. For cdk2-dependent kinase activity, the relative intensities of bands in lanes 1 through 10 were 1.0, 1.3 6 0.2, 1.7 6 0.2, 2.4 6 0.3,a 1.2 6 0.1, 1.3 6 0.2, 1.4 6 0.3,b 1.2 6 0.1, 0.9 6 0.1, and 0.9 6 0.1, respectively. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2-induced response. All data are means 6 SE for three separate (replicate) determinations. (B) Graphical representation of cdk4- and cdk2-dependent kinase activities obtained from results summarized in A.
Hormonal effects on cdk7, tyrosine phosphorylation, and cdc25A. The cyclin H/cdk7 protein or cdk-activating kinase (CAK) plays an important role in phosphorylation of both cdk2 and cdk4 at threonine-160 and threonine-172, respectively, to activate kinase activity. Although treatment of MCF-7 cells with E2 had mini-
FIG. 4. The effects of E2, TCDD, and TCDD plus E2 on p27, p21, and p53 protein levels. The whole-cell lysates (100 mg) as described in the legend to Fig. 1 were used for the assay and Western blot analyses were carried out as described under Materials and Methods. Relative p53 protein levels were not significantly changed by any treatment. Protein levels relative to controls (U) (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 1.4 6 0.2, 1.6 6 0.3, 1.6 6 0.2, 1.7 6 0.4, 2.0 6 0.5, 1.9 6 0.4, 1.4 6 0.2, 1.5 6 0.1, and 1.3 6 0.2, respectively, for p27 protein and 1.0, 1.2 6 0.2, 1.1 6 0.2, 1.1 6 0.1, 2.6 6 0.3,b 2.5 6 0.6,b 1.8 6 0.5,b 2.0 6 0.5,a 1.5 6 0.2, and 1.2 6 0.1, respectively, for p21 protein. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2-induced response. All data are means 6 SE for three separate (replicate) determinations.
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T47D human breast cancer cells and induction of several E2-responsive genes or proteins, including tissue plasminogen activator, pS2, prolactin receptor, cathepsin D, and progesterone receptor (26 –28, 46 –51). Many of the effects of ‘‘pure’’ antiestrogens ICI 164,384 and ICI 182,780, which act via complex modulation of ERmediated responses (52–55), are similar to those reported for AhR agonists in ER-positive breast cancer cells. The molecular mechanisms of cross talk between ER- and AhR-mediated responses are complex (26); however, at least one pathway has been defined for two genes, namely pS2 and cathepsin D. Inhibition of E2induced pS2 and cathepsin D gene expression by TCDD occurs through interaction of the nuclear AhR heterodimer with iDREs in 59-promoter regions of both genes (26, 27). In contrast, the more complex mecha-
FIG. 5. Effects of E2, TCDD, and TCDD plus E2 on phosphorylation of retinoblastoma protein. (A) SDS–PAGE analysis of pRB and ppRB. The whole-cell lysates (70 mg) from different treatment groups as described in the legend to Fig. 1 were analyzed on 7.5% SDS– PAGE as described under Materials and Methods. The hyperphosphorylated and hypophosphorylated forms of RB are represented as ppRB and pRB, respectively. Hypophosphorylated RB levels were not significantly altered by the various treatments, whereas hyperphosphorylated RB was significantly altered by E2, TCDD, and TCDD plus E2. Relative levels of ppRB compared to controls (U) (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 1.8 6 0.1,a 2.6 6 0.4,a 2.7 6 0.7,a 1.7 6 0.4, 1.5 6 0.3,b 1.1 6 0.3,b 1.1 6 0.2, 0.8 6 0.3, and 0.4 6 0.1, respectively. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2induced response. All data are means 6 SE for three separate (replicate) determinations. (B) Graphical representation of relative values of ppRB. Results are derived from data shown in A.
hormone-induced responses. Inhibition of tyrosine phosphorylation and the subsequent E2-mediated increases in cdk2/cdk4 forms which are not phosphorylated on tyrosine-15 may also be related to increased cdc25 phosphatase protein, which catalyzes hydrolysis of phosphorylated tyrosine residues. The results illustrated in Fig. 9 show that E2 caused 2.5-fold increase of cdc25A protein levels in MCF-7 cells after 12 h and this increase was observed 24 h after initial treatment. In contrast, TCDD alone had minimal effect on cdc25A protein and did not modulate E2-induced upregulation of this protein (Fig. 9). DISCUSSION
TCDD inhibits E2- and growth factor-induced proliferation and DNA synthesis in ER-positive MCF-7 and
FIG. 6. Effects of E2, TCDD, and TCDD plus E2 on association of p21 with cyclin D1, cdk2, and cdk4. The whole-cell lysates (350 mg) as described in the legend to Fig. 1 were immunoprecipitated by three sequential immunoprecipitations with p21 antibody as described under Materials and Methods. (A) p21– cyclin D1 association. The combined immunoprecipitates were used to investigate the association between p21 and cyclin D1 using Western blot analysis. Relative intensities of cyclin D1 protein bands compared to controls (U) (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 2.7 6 0.2,a 2.5 6 0.2,a 1.8 6 0.2,a 2.4 6 0.3, 2.2 6 0.2, 1.6 6 0.2, 1.7 6 0.2,a 1.1 6 0.1, and 0.8 6 0.1, respectively. a Significantly different (P , 0.05) from control values. (B) Dissociation of cdk2 and cdk4 from p21. The p21-immunodepleted supernatants were used to examine cdk2 and cdk4 protein levels remaining in the supernatants after p21 immunoprecipitation. Relative cdk2 protein levels in the supernatant compared to controls (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 1.5 6 0.1, 1.7 6 0.2,a 2.2 6 0.1,a 1.8 6 0.2, 1.8 6 0.3, 1.6 6 0.1, 1.3 6 0.1, 1.2 6 0.1, and 0.9 6 0.2, respectively. Relative cdk4 protein levels in the supernatant compared to controls (arbitrarily set at 1.0) were 1.0, 1.6 6 0.1,a 1.6 6 0.2,a 2.0 6 0.1,a 1.4 6 0.1, 1.3 6 0.1, 1.3 6 0.1, 1.1 6 0.1, 1.0 6 0.2, and 0.9 6 0.5, respectively. a Significantly different (P , 0.05) from control values. All data are given as means 6 SE for three separate (replicate) determinations.
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in part, to results of previous studies which reported increased cyclin D1 expression (protein and mRNA), increased cdk2- and cdk4-dependent kinase activities, and hyperphosphorylation of RB (13–15). Despite similarities between these studies, significant differences were also observed. Some of these differences may be due to cell
FIG. 7. Effects of E2, TCDD, and TCDD plus E2 on cdk-acting kinase (cyclin H/cdk7) protein levels and kinase activity. (A) Western blot analysis. The whole-cell lysates (100 mg) described in the legend to Fig. 1 were used for the assay. Cyclin H protein expression was not significantly changed by E2, TCDD, or TCDD plus E2 treatment. Relative protein levels of cdk7 in lanes 1 through 10 compared to controls (U) (arbitrarily set at 1.0) were 1.0, 1.4 6 0.1, 1.8 6 0.2,a 2.1 6 0.3,a 1.4 6 0.3, 1.5 6 0.2, 1.2 6 0.1,b 1.1 6 0.2, 1.1 6 0.2, and 0.8 6 0.1, respectively. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2-induced response. (B) Cdk7 kinase activity. Whole-cell lysates (200 mg) were incubated with 0.3 mg of anti-cdk7 antibody overnight. Fifteen microliters of protein A–agarose beads was added and incubated for an additional 3 h at 4°C. The kinase assay was carried out as described under Materials and Methods. The relative cdk7-dependent kinase activities in lanes 1 through 10 were 1.0, 1.4 6 0.1, 3.6 6 0.3,a 1.3 6 0.3, 1.4 6 0.2, 2.7 6 0.3,b 1.3 6 0.2, 2.1 6 0.1, 1.8 6 0.4, and 1.8 6 0.3, respectively. All data are means 6 SE for three separate (replicate) determinations. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2-induced response.
nisms of growth inhibition of tumors or breast cancer cells by AhR agonists are unknown (33–37, 41– 45) and were therefore investigated in this study. Modulation of cell cycle enzymes and their dependent activities by E2 in MCF-7 cells. Basal and mitogeninduced cell growth is regulated by multiple proteins which control cell cycle progression from G0/G1 to M phase and these include cyclins, cyclin-dependent kinases, and their inhibitors. Initial studies showed that in MCF-7 cells treated with 10 nM E2, there was a significant progression of the cells from G0/G1 to S phase, and this was inhibited by TCDD (Table I). Therefore, this research has focused on delineating critical E2-inducible cell cycle responses associated with G0/G1 3 S, which are inhibited via cross talk with the AhR. The effects of E2 alone observed in this study (Figs. 1–5) are comparable,
FIG. 8. Effects of E2, TCDD, and TCDD plus E2 on phosphotyrosine status of cdk2 and cdk4 proteins. (A) The whole-cell lysates (350 mg) as described in the legend to Fig. 1 were immunoprecipitated by three sequential immunoprecipitations with anti-phosphotyrosine (PY20) antibody as described under Materials and Methods. The PY20-immunodepleted supernatants were used to examine unphosphorylated-tyrosine cdk2 and cdk4 protein levels. Relative unphosphorylated-tyrosine cdk2 protein levels in the supernatant compared to controls (U) (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 1.5 6 0.3, 1.6 6 0.3, 1.8 6 0.2,a 1.5 6 0.3, 1.2 6 0.2, 1.1 6 0.2, 1.0 6 0.0, 0.9 6 0.1, and 0.7 6 0.1, respectively. Unphosphorylated-tyrosine cdk4 protein levels in the supernatant compared to controls (arbitrarily set at 1.0) in lanes 1 through 10 were 1.0, 1.8 6 0.3, 1.8 6 0.2, 2.2 6 0.2,b 1.8 6 0.1, 1.7 6 0.2, 1.8 6 0.3, 1.3 6 0.5, 1.1 6 0.1, and 1.0 6 0.3, respectively. All data were means 6 SE for three separate determinations. a Significantly different (P , 0.05) from control values; b significantly different (P , 0.05) from E2induced response. (B) Graphical representation of dephosphorylatedtyrosine cdk2 and cdk4 protein levels. Results are derived from data shown in A.
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FIG. 9. Western blot analysis of cdc25A protein levels. (A) Western blot analysis of cdc25A. Whole-cell lysates (20 mg) as described in the legend to Fig. 1 were used for the assay and Western blot analysis was carried out as described under Materials and Methods. The relative protein levels compared to controls (U) (arbitrarily set at 1.0) in lanes 1 to 10 are 1.0, 1.9 6 0.4, 2.5 6 0.2,a 2.5 6 0.3,a 2.2 6 0.4, 2.5 6 0.1, 2.6 6 0.2, 1.7 6 0.5, 1.4 6 0.3, and 1.2 6 0.0, respectively. All data are represented as means 6 SE for three determinations. a Significantly different (P , 0.05) from control values. (B) Graphical representation of cdc25A protein levels obtained from A.
maintenance prior to addition of E2. In one study (14), cells were maintained in 10 nM ICI 182,780 (a potent antimitogen) for 48 h prior to addition of 100 nM E2, whereas cells in this study were maintained in serumfree medium before adding 10 nM E2. Prall and co-workers (14) also reported that treatment of MCF-7 cells with E2 did not alter levels of immunoreactive p21 or p27 protein; however, active cyclin E–cdk2 complexes were deficient in both p21 and p27 protein complexes, and cdk2–threonine-160 phosphorylation was increased. Planas-Silva and Weinberg (15) also observed no significant changes in p21 or p27 levels in MCF-7 cells treated with E2, whereas Foster and Wimalasena (13) reported that E2 decreased p27 but not p21 protein levels in MCF-7 cells. Results of this study also show that E2 did not significantly affect immunoreactive p53, p21, or p27 levels (Fig. 4). Planas-Silva and Weinberg (15) recently showed that after treatment of tamoxifen-arrested MCF-7 cells with E2, there was rapid activation (within 6 h) of cyclin E– cdk2 complexes which was associated with redistribution of p21 from cyclin E– cdk2 to cyclin D1– cdk4 complexes. Our results showed that
treatment of MCF-7 cells with E2 followed by immunoprecipitation with p21 antibodies resulted in a timedependent increase in association of cyclin D1 with p21 (Fig. 6A), which was also observed by Planas-Silva and Weinberg (15). In contrast to their results, p21 immunodepletion studies showed that after treatment with E2, there was a significant increase of cdk2 and cdk4 proteins in the supernatant and thereby dissociation from the p21 protein complex (Fig. 6B). Thus, increased cdk4-dependent kinase activity may be due to increased cyclin D1 protein (Fig. 2) and dissociation of cdk4 from p21 (Fig. 6B), whereas E2-induced cdk2dependent kinase activity is related to increased dissociation from p21 (Fig. 6B). Another possible explanation for the coordinate increase in both cdk2- and cdk4dependent activities (Fig. 3) may be due to increased cdk7 protein and cdk7-dependent kinase activity, which activates both cdk2 and cdk4 by phosphorylation of critical threonine residues (56, 57). Results of this study show that E2 significantly induced phosphorylation of cdk2 (*cdk2, an active form of cdk2) (Fig. 1), which parallels activation of cdk7-dependent kinase activity after treatment with E2 (Fig. 7). Thus, E2induced activation of cdk7 (Fig. 7) followed by downstream activation of cdk2- and cdk4-dependent kinase activities may also be an important factor associated with the mitogenic activity of E2 in MCF-7 human breast cancer cells. Moreover, activation of cdk– cyclin complexes is dependent not only on phosphorylation of threonine-160 in cdk2 and threonine-172 in cdk4, but also on dephosphorylation of threonine-14 and tyrosine-15 in cdk2 and cdk4 (58, 59). Results of this study show that E2 increases levels of cdc25A phosphatase protein and this parallels increased levels of unphosphorylated-tyrosine cdk2 and cdk4 protein which are observed after treatment of MCF-7 with E2 (Figs. 8 and 9). These results suggest that cdc25A is another factor involved in activation of cdk2 and cdk4 kinase complexes by E2. In contrast, Planas-Silva and Weinberg (15) reported that E2 did not affect CAK activity and inactivation of cdk2 was not related to cdc25A in tamoxifen-arrested MCF-7 cells. Conflicting reports on the effects of E2 on cell cycle proteins and their activities observed in this and other studies may be due to the known interlaboratory genotypic variations of wild-type and truncated ER transcripts and altered E2 responsiveness of MCF-7 cells (60, 61). Another source of variability may be associated with different conditions for growth-arresting MCF-7 cells, which include maintenance in serum-free medium (this study and (13)) and treatment with ICI 182,780 (14) or tamoxifen (15). Effects of TCDD alone on cell cycle proteins and enzyme activities. Treatment of MCF-7 cells with TCDD alone results in minimal inhibition of cell growth (33–
ESTROGEN AND 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN IN THE CELL CYCLE
38) and changes in cell cycle distribution (Table I). Moreover, only cyclin D1 protein levels and RB phosphorylation were significantly decreased and p21 was transiently (6 h) increased after treatment of MCF-7 cells with TCDD alone. In contrast, other antiproliferative agents such as retinoids or the antiestrogens ICI 164,384/182,780 alone inhibited breast cancer cell growth and differentially modulated multiple cell cycle enzymes/proteins. For example, treatment of MCF-7 cells with ICI 182,780 decreased cdk2- and cdk4-dependent kinase activities and cyclin D1 (mRNA and protein) and increased the cdk inhibitors p27 and p21 and the proportion of hypophosphorylated RB protein (24). In T47D cells, retinoids also increased the hypophosphorylated form of RB, and changes in other cell cycle proteins were observed only after prolonged treatment, compared to the more rapid responses observed for ICI 182,780 (23). Thus, TCDD, ICI 182,780, and retinoid acid elicit common and divergent effects on cell cycle enzymes, and this is consistent with their different mechanisms of action. Antiestrogenic activity of TCDD: Inhibition of multiple E2-regulated cell cycle proteins/enzymes. It has previously been reported that TCDD inhibited E2-induced proliferation and DNA synthesis in MCF-7 and T47D cells (33–38), and the data in Table I also show that TCDD blocks the increase of cells in S phase after treatment with E2. Our results also show that cotreatment of MCF-7 cells with TCDD plus E2 resulted in significant inhibition of E2-induced hyperphosphorylation of RB and cyclin D1 proteins and cdk2-, cdk4-, and cdk7-dependent kinase activities (Figs. 2, 3, 5, and 6). Inhibition of E2-induced cdk4-dependent kinase activity by TCDD may be related to the parallel decrease of E2-induced cyclin D1 which forms the cyclin D1– cdk4 kinase complex. Inhibition of E2-induced cdk2- and cdk4-dependent kinase activities by TCDD also correlated with the significantly increased p21 levels in cotreated cells and increased association of cdk2 and cdk4 proteins with p21 as determined in the p21 antibody immunoprecipitation studies (Fig. 6). In summary, our results demonstrate that activation of cdk2- and cdk4-dependent kinase activities in MCF-7 cells after treatment with E2 is a complex process which involves multiple cell cycle proteins as depicted in Fig. 10. For example, E2 induced cyclin D1, decreased association of both cdk2 and cdk4 proteins with p21, increased cdk7-dependent kinase activity, and decreased tyrosine phosphorylation of cdk2 and cdk4 proteins, and this was accompanied by increased cdc25A phosphatase protein levels. All of these responses were accompanied by an increase in percentage of cells in S phase and a decreased percentage in G0/G1 (Table I). In contrast, TCDD reversed the effects of E2 on distribution of MCF-7 cells in the cell cycle and
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FIG. 10. Modulation of the cell cycle by E2 and TCDD plus E2. Specific steps that are induced by E2 are denoted E1. E2-induced responses inhibited by TCDD are denoted T2 and induction by TCDD alone is indicated by T1.
this was accompanied by inhibition of several other responses which were elevated after treatment with E2. Current studies are focused on further delineating the molecular mechanisms of AhR-mediated antiestrogenicity associated with inhibition of E2-responsive cell cycle proteins/enzymes and on developing AhRbased drugs for clinical treatment of breast cancer. ACKNOWLEDGMENTS S. Safe is a Sid Kyle Professor of Toxicology. We thank Betty Rosenbaum for her technical assistance.
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