ARTICLE

Effect of Long-Term Estrogen Deprivation on Apoptotic Responses of Breast Cancer Cells to 17{beta}-Estradiol

Robert X.-D. Song, Gil Mor, Fred Naftolin, Robert A. McPherson, Joon Song, Zhenguo Zhang, Wei Yue, JiPing Wang, Richard J. Santen

Affiliations of authors: R. X.-D. Song, R. A. McPherson, Z. Zhang, W. Yue, J. Wang, R. J. Santen, Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville; G. Mor, F. Naftolin, J. Song, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT.

Correspondence to: Richard J. Santen, M.D., Division of Endocrinology, University of Virginia Health Science Center, Charlottesville, VA 22908 (e-mail: rjs5y{at}virginia.edu).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: High doses of estrogen can promote tumor regression in postmenopausal women with hormone-dependent breast cancer, but the mechanism is unknown. We investigated the molecular basis of this process by using LTED cells, which were derived by growing MCF-7 breast cancer cells under long-term (6–24 months) estrogen-deprived conditions. Methods: We treated LTED and MCF-7 cells with various concentrations of 17{beta}-estradiol (estradiol) and assayed their growth by counting the cells and measured apoptosis by annexin V staining and DNA fragmentation. Using western blot analysis, we also examined the expression of the apoptosis-inducing system of the Fas death receptor protein and its ligand, FasL, in these cells. To assess the involvement of Fas and FasL in the induction of apoptosis in LTED cells, we used activating anti-Fas antibodies and the universal caspase inhibitor Z-VAD. Finally, we examined the expression of Fas protein in E8CASS and BSK3 cells, two other cell lines derived by depriving MCF-7 cells of estrogen long term, and the responses of these cells to high-dose estradiol. All statistical tests were two-sided. Results: High concentrations of estradiol (>=0.1 nM) resulted in a statistically significant, 60% reduction in the growth of LTED cells (P<.001) and in a sevenfold increase in apoptosis (P<.001) as compared with levels in vehicle-treated cells. Both LTED and MCF-7 cells expressed FasL, but only LTED cells expressed Fas. Treatment of LTED cells with 0.1 nM estradiol increased the expression of FasL. Activating anti-Fas antibodies increased apoptosis of LTED cells, which was further stimulated by estradiol. Z-VAD blocked estradiol-induced apoptosis. E8CASS cells, which express Fas protein, but not BSK3 cells, which do not, also responded to 0.1 nM estradiol by increasing apoptosis. Conclusion: Tumor regression induced by high-dose estrogen therapy in postmenopausal woman may result from estrogen activation of Fas-mediated apoptosis.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Traditional hormonal therapies for women with breast cancer have included hormone-ablative and hormone-additive strategies. Initial clinical approaches with hormonal ablation included oophorectomy, adrenalectomy, and hypophysectomy. Medical approaches with antiestrogens and aromatase inhibitors subsequently superseded the use of surgery (1). Paradoxically, hormone-additive therapy with high-dose estrogen also causes tumor regression in postmenopausal women with estrogen receptor (ER)-positive breast cancer (2). The duration of the postmenopausal period is one of the crucial factors affecting the success of this therapy. For example, when the synthetic estrogen diethylstilbestrol (DES) was used at 150 mg per day, women who had experienced the onset of menopause less than 1 year before therapy did not respond to DES, women within 5 years of menopause experienced a 7.9% objective response rate, and women who had reached menopause more than 10 years earlier experienced a 22% response rate (3). These clinical observations suggest that long-term exposure to low levels of estrogen might alter the responsiveness of breast cancer cells to additive estrogen administration.

Once tamoxifen was developed, in the 1970s, the use of high-dose estrogen as hormone-additive treatment of breast cancer fell out of favor, and tamoxifen became standard first-line therapy. This change in practice resulted from a clinical trial with a 4-year follow-up (4) that demonstrated that postmenopausal women with advanced breast cancer tolerated tamoxifen statistically significantly better than they tolerated DES (P = .02) while experiencing objective response rates that were not statistically significantly different (33% for tamoxifen and 42% for DES). A recent report with a 20-year follow-up observation of the same trial participants (5), however, revealed two surprising findings. First, DES-treated patients survived longer than patients receiving tamoxifen, with 35% of the DES-treated group and just 16% of the tamoxifen-treated group alive at 5 years (P = .03). Second, 30% of patients who crossed over to the DES arm from the tamoxifen arm responded to DES. These findings suggest that the mechanistic effects of estrogen-additive therapy differ from those of tamoxifen.

To investigate the mechanism of estrogen-additive therapy, a cellular model is needed. MCF-7 breast cancer cells deprived of estrogen long term by growing them in medium treated with activated charcoal to remove substantial amounts of estrogen, thus mimicking the hormonal milieu of breast cancer cells in postmenopausal women (6), initially stop growing but resume rapid proliferation after a period of 3–5 months (7,8). The growth of these long-term estrogen-deprived (LTED) cells in vitro can, like that of breast tumors of postmenopausal women, be inhibited by antiestrogens, suggesting that small amounts of estrogen may promote this regrowth. In vivo, LTED xenografts in nude mice also grow in response to lower amounts of estradiol than do parental MCF-7 cells (6). In contrast, higher concentrations of estradiol partially inhibit the growth of LTED xenografts, compared with its stimulatory effects on MCF-7 xenografts (6). The mechanism for the inhibitory effect of estradiol on LTED cell growth is not clear. On the basis of these observations, we believe that LTED cells can serve as an in vitro model suitable for studying the mechanism of high-dose estrogen-induced cancer cell regression in postmenopausal women.

Data obtained during characterization of LTED cells in our laboratory have suggested a possible mechanism by which high-dose estrogen induces tumor regression. Many proteins, including c-Myc, E2F1, Ras, and mitogen-activated protein (MAP) kinase, are expressed at higher levels in LTED cells than in MCF-7 cells (9,10). These proteins can exert bifunctional effects, stimulating proliferation on the one hand or inducing apoptosis on the other, depending on the conditions (11,12). Although estradiol usually inhibits apoptosis, some studies (13,14) provide evidence for a paradoxical induction of apoptosis by estradiol. Because tumor growth depends on the suppression of apoptosis as well as stimulation of cell proliferation, it is possible that high-dose estrogen-induced breast tumor regression might be due to an enhancement of apoptotic cell death.

We further postulated that the Fas/Fas ligand (FasL) pathway might be involved in the induction of apoptosis by high-dose estradiol (15). Activation of the Fas and FasL pathway can be highly regulated to serve as a means of modulating cell apoptotic processes (15). Fas (also known as CD95 or Apo-1) is a type I transmembrane protein of the tumor necrosis factor (TNF) receptor family, and FasL (also known as CD95L or Apo-1L) belongs to the family of TNF-related cytokines (15). The majority of breast carcinomas express FasL protein (16), but the expression of Fas protein is more heterogeneous in both benign and malignant breast tissues and cell lines (17,18). An ER response element (ERE) has been identified in the promoter region of the FasL gene (19). Enhanced expression of FasL induced by estradiol is a critical factor in the breast tumor's ability to evade the immune system because it leads to apoptosis of Fas-bearing lymphocytes (19).

The goal of this study was to investigate whether high-dose 17{beta}-estradiol (estradiol) causes breast tumor regression in postmenopausal women by a mechanism involving promotion of apoptotic cell death. To that end, we examined in a model system whether long-term estrogen deprivation can "prime" MCF-7 cells to the apoptotic effect of estradiol and lead to tumor regression. We also analyzed the effect of long-term estrogen deprivation on Fas and FasL protein expression, examined whether treatment of LTED cells with high-dose estradiol affects Fas or FasL expression, and assessed whether Fas/FasL expression is associated with apoptotic cell death of LTED cells. Finally, we repeated some of these analyses with E8CASS and BSK3 cells, two other cell lines that are adapted to growth long term under low-estrogen conditions.


    MATERIALS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Materials

Tissue culture supplies were obtained from Fisher Scientific (Pittsburgh, PA). Iscove's modified Eagle's medium (IMEM; zinc option Richter's modification) with or without phenol red and fetal bovine serum (FBS) were obtained from Life Technologies, Inc. [GIBCO BRL], Rockville, MD. 17{beta}-Estradiol (estradiol) was obtained from Steraloids, Wilton, NH. ICI 182780 was from AstraZeneca, Inc., Wilmington, DE. Both estradiol and ICI 182780 were dissolved in 100% ethanol. Z-VAD was purchased from Calbiochem, San Diego, CA, and was dissolved in dimethyl sulfoxide (Sigma Chemical Co., St. Louis, MO). All other chemicals were of reagent grade and were obtained from either Sigma Chemical Co. or Fisher Chemical Co. For the preparation of dextran-coated charcoal-stripped FBS (DCC-FBS), the FBS was heat-inactivated at 56 °C for 30 minutes and then mixed with Norit A-activated charcoal (4 mg/mL) and dextran T-70 (0.4 mg/mL; Pharmacia, Piscataway, NJ) overnight at 37 °C. The serum was then centrifuged at 33 000g for 60 minutes at 4 °C. The centrifugation was repeated three times. The supernatant was filtered first through a 0.22-µm filter (Nalgene, Rochester, NY) and then through a 0.1-µm filter (Millipore Corp., Bedford, MA) to remove fine charcoal particles that could contain residual estrogen (6).

Cell Culture Conditions

LTED cells were derived by growing MCF-7 cells for 6 months to 2 years in estrogen-depleted medium (6). The characteristics of the cells have been reported previously (7,9). E8CASS and BSK3 cells were from of Dr. C. Sonnenschein, Tufts University, Boston, MA, and Dr. B. S. Katzenellenbogen, University of Illinois, Urbana–Champaign, IL, respectively. Both cell lines were developed from MCF-7 cells by long-term estrogen deprivation (20,21). LTED, E8CASS, and BSK3 cells were maintained routinely in phenol red-free IMEM supplemented with 5% DCC-FBS, and MCF-7 cells were maintained in phenol red-containing IMEM supplemented with 5% FBS. The cells were cultured in humidified 95% air and 5% CO2 at 37 °C. For all experiments unless otherwise specified, the medium was changed into phenol red-free IMEM containing 1% DCC-FBS 1 day after cells were seeded. Then 24 hours later, the medium was changed into fresh phenol red-free IMEM containing 1% DCC-FBS, and the cells were treated with different agents as indicated.

Assessment of Cell Growth

Cells growing in six-well plates were treated with estradiol at concentrations ranging from 0.00001 nM to 10 nM in individual experiments. After growth for the times specified in the figures, cells were rinsed once with 0.9% saline and then lysed in ZAP buffer (i.e., 0.01 M HEPES [pH 7.2], 1.5 mM MgCl2, and 0.13 M ethylhexadecyldimethylammonium bromide [ZAP; Kodak, Rochester, NY]). The released nuclei in 1 mL of ZAP buffer were mixed with 9 mL of isoton II diluent (catalog No. 23–375–212; Coulter Corp., Miami, FL) and counted with a model Z1 Coulter counter (Coulter Corp.).

Assessment of Apoptosis

Apoptosis was assessed by three methods (22): annexin V staining, which detects an early event in apoptosis, and DNA fragmentation and morphology, which detect later events. Annexin V staining was conducted with the use of a fluorescein isothiocyanate (FITC)-labeled annexin V apoptosis kit (Sigma Chemical Co.). In brief, MCF-7 and LTED cells were plated in 24-well plates at a density of 1 x 104 cells per well. On the next day, the medium was replaced with fresh IMEM containing 1% DCC-FBS for both MCF-7 and LTED cells. The cells were then exposed to 0.1 nM estradiol or ethanol as vehicle control for 12 hours. At the end of the experiment, FITC-labeled annexin V was added to the culture medium to a final concentration of 1 µg/mL in the presence of 0.2 mM CaCl2, and the cells were incubated for 10 minutes at 37 °C. Apoptotic cells were identified by direct visualization of green plasma membrane staining under the fluorescence microscope. Generally, more than 10 representative fields of each well were analyzed. Both light and fluorescent images were taken for further analysis.

For measurements of DNA fragmentation, LTED cells were treated with 0.1 nM estradiol or ethanol as vehicle control for a 3-day period. DNA fragmentation was quantified with a commercially available kit (the Cell Death Detection ELISA [i.e., enzyme-linked immunosorbent assay] Plus Kit; Roche Molecular Biochemicals, Indianapolis, IN). Cell extracts of each sample were prepared and analyzed according to the manufacturer's protocol and were read on a plate reader at 405 nm. The quantity of cleaved DNA measured by ELISA as an indicator of apoptosis was normalized to the number of cells present. In some experiments, the antiestrogen ICI 182780 or the pan-caspase inhibitor Z-VAD was included. For these experiments, cells were pretreated with different concentrations of the inhibitors for 30 minutes and then treated with estradiol for the indicated time.

In some cases, the DNA fragments were analyzed by gel electrophoresis with the Apoptotic DNA Ladder Kit (Roche Molecular Biochemicals). Briefly, after a 3-day treatment with estradiol or vehicle, LTED cells were washed once in phosphate-buffered saline and resuspended in 400 µL of the DNA binding/lysis buffer included in the kit. The lysate was applied to glass fiber fleece in a filter column. The column was washed three times with the washing buffer provided in the kit, and the DNA specifically bound to the column was eluted with the elution buffer provided in the kit according to the manufacturer's directions. Three micrograms of the eluted DNA from each treatment was separated on a 0.5% agarose gel containing ethidium bromide, and the DNA was visualized with the use of UV light.

For morphologic studies of apoptosis, LTED and MCF-7 cells were treated with 0.1 nM estradiol or vehicle for a 3-day period. Cell morphology was evaluated by phase-contrast light microscopy (Nikon, Melville, NY) on days 1–3 for the LTED cells and on day 3 only for MCF-7 cells.

Western Blot Analysis of Fas and FasL

Proteins were extracted from cells with the use of Trizol reagent (Life Technologies, Inc.) according to the manufacturer's instructions. The extract was analyzed for protein concentration with a Lowry protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein (25 µg) from each sample were separated on sodium dodecyl sulfate containing 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes (MSI, Westborough, MA). Membranes were blocked in 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and incubated with an anti-FasL monoclonal antibody (1 : 1000 dilution of clone 33; Transduction Laboratory, Lexington, KY) or an anti-Fas monoclonal antibody (1 : 500 dilution of B-10; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. The immunoblots were then washed three times with TBS-T buffer, incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (horse anti-mouse; Vector Laboratories, Inc., Burlingame, CA), and developed with the HRP substrate kit (Vector Laboratories, Inc.).

Fas-Induced Apoptosis

To study Fas protein-induced apoptosis, we used an activating anti-human Fas monoclonal antibody (MAB142; R&D, Minneapolis, MN). LTED cells were plated at a density of 2000 cells per well in IMEM containing 5% DCC-FBS in 96-well plates. Two days later, the medium was replaced with 200 µL of fresh 1% DCC-FBS IMEM. The cells were then treated with the antibody at 500 ng/mL or with 0.1 nM estradiol for 3 days. Mouse immunoglobulin G antibody (Santa Cruz Biotechnology) was used as a control. Apoptosis was assayed on the basis of DNA fragmentation with the use of the Cell Death Detection ELISA Plus Kit.

Statistical Analysis

Statistical differences between means for cell counts and ELISAs were determined with two-tailed Student's t tests. Whenever the group means were statistically significantly different, 95% confidence intervals (CIs) for the means were constructed. All P values were two-sided.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Effects of Estradiol on Growth of LTED and MCF-7 Cells

To study the effect of estradiol on the growth of cells that had been subjected to long-term estrogen deprivation, we initially plated both LTED and MCF-7 cells at 1 x 105 cells per well and counted them at day 4 after they had been treated with different concentrations of estradiol or vehicle (ethanol). For LTED cells, the cell number after vehicle treatment was 1.4 x 106 cells per well (95% CI = 1.21 to 1.59 x 106 cells per well). Treatment of the LTED cells with estradiol at 0.1 nM and 10 nM resulted in lower cell numbers, 0.65 x 106 cells per well (95% CI = 0.56 to 0.74 x 106 cells per well) and 0.6 x 106 cells per well (95% CI = 0.51 to 0.69 x 106 cells per well), respectively (Fig. 1Go). Compared with vehicle treatment, the above two concentrations of estradiol resulted in 60% fewer cells, a reduction that was statistically significant (P<.001). Estradiol at a concentration of 10 nM did not produce a further reduction in cell number as compared with 0.1 nM estradiol. Unlike LTED cells, which grew independently of estradiol, MCF-7 cells grew much more slowly in estrogen-depleted medium, with 0.33 x 106 cells per well in vehicle-treated wells (95% CI = 0.27 to 0.39 x 106 cells per well) (Fig. 1Go). Estradiol increased MCF-7 cell growth in a dose-dependent fashion, with 10 nM estradiol maximally stimulating cell growth, to 1.1 x 106 cells per well (95% CI = 0.91 to 1.3 x 106 cells per well, P<.001), which represents a threefold increase over the growth of vehicle-treated MCF-7 cells. Together, these results indicate that higher concentrations of estradiol decreased LTED cell growth but increased MCF-7 cell growth.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1. Effect of 17{beta}-estradiol (E2) on numbers of LTED and MCF-7 cells. Cells were grown as described in the "Materials and Methods" section and were then treated with E2 at the indicated concentrations or with vehicle (ethanol) for 4 days. Cell numbers were counted with a Coulter counter. 1E-05 is a designation for 0.00001 nM E2. Points represent the means of cell count for one representative experiment run in triplicate. Error bars represent 95% confidence intervals of the means.

 
Effects of Estradiol on Apoptosis of LTED and MCF-7 Cells

Because a decrease in cell number can result from an increase in apoptosis, we investigated whether estradiol-induced cell growth arrest in LTED cells might be due to increased apoptosis. The process of apoptosis has an early phase as well as a late phase; the early phase can be detected by annexin V staining, and the late phase can be detected by DNA fragmentation and morphology. Fig. 2, AGo, shows that, after 12 hours of vehicle treatment of LTED cells, annexin V staining was not detectable. Treatment with estradiol at 0.1 nM for 12 hours strongly increased annexin V staining of LTED cells. No apoptotic cells were observed in either vehicle-treated or estradiol-treated MCF-7 cells (Fig. 2, AGo).






View larger version (270K):
[in this window]
[in a new window]
 
Fig. 2. 17{beta}-Estradiol (E2) effect on apoptosis of LTED and MCF-7 cells. A) Effect of E2 on annexin V membrane binding. Cells were treated with vehicle (controls) or with 0.1 nM E2 for 12 hours. Apoptosis was observed by annexin V-FITC binding to the cell membrane as described in the "Materials and Methods" section. Each panel shows a companion fluorescent image (left) and bright-field image (right). Bar = 10 µm. B) Concentration dependence of E2's effect on apoptosis in LTED (left panel) and MCF-7 (right panel) cells. Cells were treated with the indicated concentrations of E2 for 3 days (1E-05 is a designation for 0.00001 nM E2). DNA fragmentation of apoptosis was assayed by the enzyme-linked immunoassay method as described in the "Materials and Methods" section. The data represent the fold increase of the means over the control (i.e., vehicle-treated cells) for an experiment run in triplicate. Error bars represent 95% confidence intervals. C) Analysis of DNA fragmentation by agarose gel electrophoresis. LTED cells were treated with vehicle or 0.1 nM E2 for 3 days. Genomic DNA was extracted as described in the "Materials and Methods" section and analyzed on a 0.5% agarose gel. DNA samples loaded on gel are from vehicle-treated LTED cells (lane 2) and E2-treated LTED cells (lane 3). Lane 1 was loaded with 1-kilobase DNA ladder as a molecular weight control. One representative experiment is shown. D) Effect of E2 on the morphology of LTED and MCF-7 cells. Cells were treated with vehicle or with 0.1 nM E2 for 3 days. Photographs were taken with a phase-contrast microscope every day for LTED cells and on day 3 for MCF-7 cells. Bar = 100 µm.

 
To confirm the effect of estradiol on LTED cell apoptosis, we analyzed DNA fragmentation by an ELISA. Both LTED and MCF-7 cells were treated with different concentrations of estradiol for 3 days (Fig. 2, BGo). A statistically significant, sevenfold increase in apoptosis in LTED cells (95% CI = 5.5-fold to 8.5-fold, P<.001) relative to vehicle-treated cells was observed at an estradiol concentration of 0.1 nM (Fig. 2, B; left panelGo). It is interesting that MCF-7 cells cultured in the absence of estradiol showed a detectable basal level of apoptosis. However, apoptosis of MCF-7 cells was diminished in the presence of estradiol, with maximum effect at 10 nM, the highest concentration tested (Fig. 2, B; right panelGo).

Agarose gel electrophoresis was used to confirm the DNA fragmentation as further evidence of estradiol-induced apoptosis in the LTED cells (Fig. 2, CGo). DNA isolated from the vehicle-treated LTED cells yielded bands only in the high-molecular-weight region. In contrast, DNA cleavage was evident after 3 days of treatment with 0.1 nM estradiol. The DNA fragmentation appears as a smear on the gel rather than as characteristic nucleosomal laddering. It has been noted previously (23,24) that the DNA of MCF-7 cells undergoing apoptosis does not exhibit a characteristic nucleosomal laddering pattern because of their lack of caspase 3. DNA in MCF-7 cells, when undergoing apoptosis, is cleaved into fragments of progressively smaller sizes, resulting in a smear rather than in discrete nucleosome laddering. This finding may explain the lack of discrete nucleosomal bands in DNA from estradiol-treated LTED cells.

Late-stage apoptosis can be detected not only by DNA fragmentation but also by morphologic changes, such as cell rounding and detachment. Using phase-contrast microscopy, we observed time-dependent changes in LTED cell morphology after estradiol treatment. Vehicle-treated LTED cells displayed a normal morphology, with a monolayer appearance, from day 1 to day 3 (Fig. 2, DGo). In contrast, treatment with 0.1 nM estradiol resulted in cellular morphologic changes reminiscent of the apoptotic phenotype beginning 2 days after estradiol was added (Fig. 2, DGo). These changes included the presence of phase-bright cell bodies, shrunken cytoplasm, and an increased number of floating cells. Unlike LTED cells, vehicle-treated MCF-7 cells for 3 days showed normal cell morphology, with a few floating cells that might have accounted for the increased basal levels of apoptosis detected by the DNA fragmentation assay. MCF-7 cells treated with estradiol did not show obvious morphologic changes compared with vehicle-treated cells at day 3 (Fig. 2, DGo). Taken together, these results demonstrate that LTED cells are sensitive to the apoptotic effects of estradiol, whereas MCF-7 cells seem to be protected from apoptosis by estradiol.

Role of ER in Estradiol Effect on Apoptosis

To determine if the ER is involved in the estradiol-induced apoptosis of LTED cells, we blocked ER function with increasing concentrations of the pure antiestrogen ICI 182780 and examined the effect on apoptosis (Fig. 3Go). Pretreatment of LTED cells with ICI 182780 for 30 minutes blocked estradiol-induced apoptosis in a concentration-dependent manner, with a maximum effect at 100 nM ICI 182780, the highest concentration tested. It is interesting that ICI 182780 alone, which induces apoptosis in MCF-7 cells (25,26), had little effect on apoptosis of LTED cells. Taken together, these data suggest that ERs are involved in estradiol actions on promotion of apoptosis of LTED cells.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3. Estrogen receptor involvement in 17{beta}-estradiol (E2) action on apoptosis. LTED cells were pretreated with different concentrations of the ICI 182780 (ICI) for 30 minutes and then treated with vehicle (open bars) or with 0.1 nM E2 (solid bars) for 3 days. DNA fragmentation, a measure of apoptosis, was assessed by enzyme-linked immunosorbent assay. The data represent the means of the fold induction over the vehicle-treated control for one of the experiments run in triplicate. Error bars represent 95% confidence intervals.

 
FasL and Fas Protein Expression in LTED and MCF-7 Cells

The Fas/FasL signaling pathway plays an important role in the initiation of apoptosis (27). We have demonstrated previously that FasL, but not Fas, is expressed in MCF-7 cells and that FasL messenger RNA and protein levels are increased by estradiol treatment (19). In the present study, Fas protein expression and FasL protein expression were further assessed by western blot analysis in both LTED and MCF-7 cells. FasL was clearly detectable in LTED and MCF-7 cells (Fig. 4, AGo). By contrast, Fas was detectable in LTED cells only. MCF-10A cells were used as a negative control for FasL and as a positive control for Fas proteins (28). To examine the effects of estradiol on the expression of FasL and Fas in LTED cells, we treated LTED cells with 0.1 nM estradiol for varying times. Compared with treatment with vehicle alone, estradiol treatment increased FasL protein expression in LTED cells by twofold (95% CI = 1.3-fold to 2.7-fold) at 24 hours and threefold (95% CI = 2.2-fold to 3.9-fold) at 48 hours (Fig. 4, BGo) and in MCF-7 cells (data not shown). Expression of Fas protein in LTED cells was not affected by estradiol treatment.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4. Western blot analysis of Fas and Fas ligand (FasL) expression. LTED, MCF-7, and MCF-10A cells (A) were cultured in Iscove's modified Eagle's medium containing 1% dextran-coated charcoal-stripped fetal bovine serum. LTED cells (B) were treated with vehicle (i.e., control) or with 0.1 nM 17{beta}-estradiol (E2) for the times indicated. Cells were then extracted with Trizol reagent, and the expression of Fas and FasL was measured by western blot analysis. The molecular masses of Fas (46 kilodaltons [kDa]) and of FasL (30 kDa) are indicated. Lower panels show quantitative densitometric scanning results from three independent experiments. The means for Fas protein expression are shown in open bars and for FasL in solid bars. Error bars indicate 95% confidence intervals.

 
Functional Involvement of Fas and Caspase Pathways in LTED Cells

LTED cells express both FasL and Fas proteins, whereas MCF-7 cells have FasL but no detectable Fas. Accordingly, we hypothesized that the increased Fas protein in LTED cells might provide sufficient receptor for FasL to function. The estradiol-induced increments in FasL would then lead to LTED cell apoptosis. To explore the functional involvement of the Fas/FasL pathway in the estradiol-induced apoptosis of LTED cells, we evaluated the sensitivity of both LTED and MCF-7 cells to an activating anti-Fas monoclonal antibody. Treatment of cells with the anti-Fas antibody at 0.5 µg/mL or with estradiol at 0.1 nM stimulated LTED cell apoptosis to levels twofold (95% CI = 1.7-fold to 2.3-fold) and threefold (95% CI = 2.4-fold to 3.6-fold) over those in vehicle-treated cells, respectively (Fig. 5, A; left panelGo). Furthermore, a combination of anti-Fas antibody with estradiol had an additive effect on apoptosis of LTED cells (Fig. 5, A; left panelGo), suggesting that a common signaling pathway is activated, although the use of anti-Fas blocking antibodies will be necessary to confirm this result. MCF-10A cells, a normal human mammary cell line that expresses Fas protein (17), were used as a positive control; these cells responded to the antibody with increased apoptosis (Fig. 5, A; right panelGo). As in our previous (17) and current studies, MCF-7 cells were resistant to the effects of the antibody on apoptosis, which is consistent with their lack of Fas protein expression (Fig. 5, A; right panelGo).




View larger version (40K):
[in this window]
[in a new window]
 
Fig. 5. Effects of activating anti-Fas antibody and the pan-caspase inhibitor Z-VAD on 17{beta}-estradiol (E2)-induced apoptosis. A) Effect of activating anti-Fas antibody on E2-induced apoptosis of LTED cells. LTED cells (left panel) were treated with 0.5 µg/mL monoclonal anti-Fas (anti-Fas mAb) or with 0.5 µg/mL isotype-matched control monoclonal antibody (control mAb) in the presence or absence of 0.1 nM E2. MCF-10A and MCF-7 cells (right panel), treated with control mAb or anti-Fas mAb, were used as positive and negative controls for LTED cells, respectively. B) LTED cells were pretreated with various concentrations of Z-VAD for 30 minutes and then treated with vehicle (open bars) or 0.1 nM E2 (solid bars) for 3 days. In both sets of experiments, DNA fragmentation, a measure of apoptosis, was assessed by enzyme-linked immunosorbent assay. The data represent the increased apoptosis as fold of the vehicle-treated control from an experiment performed in triplicate. Error bars represent 95% confidence intervals.

 
Stimulation of the Fas/FasL pathway induces caspase activation. Accordingly, we determined the effect of the pan-caspase inhibitor Z-VAD on estradiol-induced apoptosis of LTED cells. Fig. 5, BGo, shows that Z-VAD blocked the apoptosis induced by 0.1 nM estradiol in a concentration-dependent manner. This result indicates that caspase activation is necessary for estradiol-induced apoptosis of LTED cells and provides further evidence for the role of Fas/FasL in apoptotic signaling in these cells.

Effect of Estradiol on Other MCF-7 Cells Adapted to Long-Term Estrogen Deprivation

LTED cells are not the only MCF-7 cells adapted to growth under long-term estrogen-deprived conditions. We tested two other such cell lines, E8CASS (20) and BSK3 (21), to determine whether these lines respond to estradiol similarly to LTED cells. As shown in Fig. 6, AGo, estradiol at 0.1 nM produced a statistically significant induction of apoptosis in both LTED and E8CASS cells relative to vehicle-treated cells. The increase was 5.5-fold (95% CI = 4.2-fold to 6.8-fold; P<.001) in LTED cells and 8.2-fold (95% CI = 6.4-fold to 10-fold, P<.001) in E8CASS cells. Like LTED cells, E8CASS cells responded to estradiol treatment with a statistically significant decrease in cell number (95% CI = 0.89 to 1.21 x 106 cells per well; P<.001) over a 4-day period (Fig. 6, BGo). It is interesting that BSK3 cells did not respond to estradiol by either inducing apoptosis or altering cell growth (Fig. 6, A and BGo). Further studies revealed that the basal level of Fas protein in E8CASS cells was increased, whereas that of BSK3 was not (Fig. 6, CGo).




View larger version (68K):
[in this window]
[in a new window]
 
Fig. 6. 17{beta}-Estradiol (E2) effects on MCF-7 breast cancer cells and three derivative cell lines (i.e., LTED, E8CASS, and BSK3) created by long-term estrogen deprivation. Cells were treated with vehicle (open bars) or with 0.1 nM E2 (solid bars). DNA fragmentation, a measure of apoptosis, was determined after 3 days by enzyme-linked immunoabsorbent assay (A), and the cell number was determined after 4 days by a Coulter counter (B). The expression levels of Fas and Fas ligand (FasL) in untreated cells were determined by immunoblot analysis (C). The data in panel A represent the means of fold induction over the vehicle-treated control for each cell type, and the data in panel B represent the mean cell counts for each treatment. All experiments were performed three separate times, with each in triplicate, and one representative experiment is shown. Error bars represent 95% confidence intervals.

 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hormone-additive therapy with high-dose estradiol induces tumor regression in postmenopausal women with ER-positive breast cancer, a paradoxical finding given that hormone-ablative therapy can also be used to treat breast cancer. The frequency of responses to hormone-additive therapy increases proportionately with duration of the postmenopausal state. We used LTED cells, which are MCF-7 breast cancer cells that had been deprived of estrogen for a long period in culture, to investigate the hypothesis that long-term estrogen deprivation initiates an adaptive process that allows a high dose of estradiol to induce cell apoptosis and, presumably, tumor regression. To our knowledge, our results demonstrate for the first time that high doses of estradiol, mediated by the ER, produce a statistically significant decrease in cell growth and increase in apoptosis in LTED cells. The molecular mechanism responsible for the induction of apoptosis is likely to involve the increased amount of Fas protein that appears in response to long-term estrogen deprivation. Furthermore, activation of caspases via the Fas/FasL pathway appears to be involved in estradiol promotion of apoptosis in LTED cells. These observations provide a potential explanation for the antitumor effects of additive estrogen therapy in postmenopausal women.

The process of adaptation of breast cancer cells to long-term estrogen deprivation involves activation of several critical regulatory factors, such as c-Myc, c-Myb, MAP kinase, E2F1, and Ras (6,9,10). These proteins can mediate the action of hormones and growth factors by exerting dual but opposing effects on cell proliferation and apoptotic cell death (2932). We demonstrated previously that LTED cells express substantially elevated c-Myc protein levels compared with parental MCF-7 cells (9). It has been reported that c-Myc can sensitize T lymphocytes to the apoptotic effects of Fas/FasL pathway activation by removing Saf (suppressor of apoptosis by Fas), a factor normally associating with Fas (3335). Therefore, it seems likely that the elevated c-Myc protein in LTED cells is involved in the regulation of Fas/FasL activation in the apoptosis-promoting action of estradiol.

Paradoxical induction of apoptosis by estrogen has been described previously under several additional circumstances. For example, estradiol induced apoptosis in MCF-7 breast cancer xenografts adapted to conditions of long-term estradiol blockade with tamoxifen (14,36). Estrogen also induces apoptosis in rat embryo fibroblasts (Rat1 cells) stably transfected with ER{alpha} (13) and in MCF-7 cells stably transfected with Raf-1 (37). In addition, the response of neurons to estradiol depends on the ER subtype of the cells: Cells with ER{beta} undergo apoptosis, whereas cells with ER{alpha} are protected from apoptosis (28). Although the mechanisms underlying these effects of estradiol are not clear, apoptosis mediated by ER{beta} requires FasL (28). In addition, an increase in Fas protein expression is associated with the ovariectomy-induced regression of the vaginal mucosa (38). Similarly, apoptosis is seen in Fas-transfected L1210 T lymphocytes but not in parental cells, in which Fas expression is low (39). The process of tissue remodeling in the mammary gland after termination of lactation also involves the Fas/FasL pathway (17). FasL activation by estradiol in MCF-7 cells results in the apoptosis of Fas-bearing lymphocytes, thereby allowing tumors to escape immune detection (19). It is interesting that both LTED and MCF-7 cells express FasL, but only LTED cells have the requisite receptor (i.e., Fas) to allow apoptosis to occur in response to activation of FasL by estradiol. We are currently investigating the mechanism by which long-term estrogen deprivation increases Fas expression.

Two other cell lines that were developed from MCF-7 cells by long-term estrogen deprivation, E8CASS (20) and BSK3 (21), differed in their response to estradiol, with E8CASS cells, but not BSK3 cells, undergoing apoptosis in response to estradiol. This result is partially due to an increase in Fas protein expression in E8CASS cells but not in BSK3 cells. E8CASS cells have also been demonstrated to undergo growth arrest in response to estrogen treatment (20). The differences in the responses to estradiol in the three cell lines are currently unknown, but they may reflect differences in their derivation. E8CASS cells were developed by selection of cloned cells after long-term estrogen deprivation (20), which may explain why their responses to estrogen were stronger than those of LTED cells. On the other hand, BSK3 cells were developed without clonal selection. Alternatively, the timing of adaptation to long-term estrogen deprivation might explain the differences.

Our results demonstrate hormonal induction of apoptosis in MCF-7 derivative cells, but they do not provide evidence that this phenomenon may be generalizable to other breast cancer model systems or human tumors. However, analogous findings have been reported in the commonly studied LNCaP prostate cancer cell line (40). When grown and serially passaged in androgen-containing culture medium, androgens stimulate proliferation and inhibit apoptosis in these cells. These experiments involve acute step- down into androgen-depleted medium and then examination of the effects of exogenously added androgen. However, when LNCaP cells are adapted to long-term androgen deprivation by serial passage in androgen-depleted medium, they undergo apoptosis in response to the addition of the androgenic steroid R-1881 (40). The fact that prostate cancer cells and breast cancer cells respond similarly to long-term hormone deprivation supports the possibility that this may be a common biologic response in hormone-responsive tissue.

The rate of tumor growth or regression represents a dynamic balance between the number of new cells generated and cells undergoing apoptotic death. Our results provide strong evidence that high-dose estradiol stimulates apoptosis in LTED and E8CASS cells. Thus, apoptosis can contribute to the diminution of tumor growth. However, we have not presented data regarding generation of new cells. Our preliminary findings (unpublished data) indicate that estradiol treatment of LTED cells reduces incorporation of tritiated thymidine into DNA. Therefore, reduced cell proliferation probably contributes to the reduction in cell number induced by estradiol. Accordingly, further studies are required to precisely quantify the relative contributions of apoptosis and decreased mitosis in reducing cell number. In addition, the molecular mechanisms responsible for estradiol-induced reduction of DNA synthesis require clarification.

Induction of apoptosis by estradiol has substantial clinical relevance, provided that our findings are confirmed in women with postmenopausal breast cancer. Hormone-additive therapies with DES or ethinyl estradiol promote tumor regression in postmenopausal women with breast cancer. Moreover, the effects of DES show a clear dose response. In a multicenter trial, daily treatment with 1.5 mg of DES (a dose that is approximately threefold higher than the estrogen replacement level) induced objective responses in 10% of patients (3). Increasing the daily dose to 15, 150, or 1500 mg produced a linear increase in the response rate, to a maximum of 24.5% at the 1500-mg dose level (3). These response rates would probably have been greater if patients had been selected on the basis of having ER-positive cancer. Responses also increased linearly as a function of duration of the postmenopausal period. Demonstration that these effects are a result of apoptosis would introduce a new concept of hormonal therapy for breast cancer, namely, the hormonal induction of apoptosis. Measurement of the rate of apoptosis would then allow characterization of the dose–response characteristics of estrogen. More speculatively, regimens with sequential use of antiestrogens and additive estrogen might turn out to be useful in the treatment of breast cancer in postmenopausal women.

In summary, our data on breast cancer cells adapted to growth under conditions of estrogen deprivation demonstrate that additive estrogen causes a paradoxical reduction in breast cancer cell number in vitro that is associated with enhancement of apoptosis. The induction of apoptosis by estradiol in the LTED cells may be mechanistically related to the presence of the Fas receptor and involve activation of the Fas/FasL pathway. These findings suggest that long-term estradiol deprivation sensitizes cells to the proapoptotic effects of high doses of estradiol. These results and other recent clinical data (35) reporting increased survival in patients treated with DES versus tamoxifen emphasize the need to reconsider use of additive estrogen therapy in breast cancer patients and to conduct further mechanistic studies of the effects of this therapy.


    NOTES
 
Supported by Public Health Service (PHS) grant R01CA165622 (National Cancer Institute) (to R. J. Santen) and PHS grant R01HD37137 (National Institute of Child Health and Human Development) (to G. Mor), National Institutes of Health, Department of Health and Human Services.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

1 Santen RJ, Worgul TJ, Samojlik E, Interrante A, Boucher AE, Lipton A, et al. A randomized trial comparing surgical adrenalectomy with aminoglutethimide plus hydrocortisone in women with advanced breast cancer. N Engl J Med 1981;305:545–51.[Abstract]

2 Kennedy BJ. Massive estrogen administration in premenopausal women with breast cancer. Cancer 1962;15:641–8.[Medline]

3 Carter AC, Sedransk N, Kelley RM, Ansfield FJ, Ravdin RG, Talley RW, et al. Diethylstilbestrol: recommended dosages for different categories of breast cancer patients. Report of the Cooperative Breast Cancer Group. JAMA 1977;237:2079–85.[Abstract]

4 Ingle JN, Ahmann DL, Green SJ, Edmonson JH, Bisel HF, Kvols LK, et al. Randomized clinical trial of diethylstilbestrol versus tamoxifen in postmenopausal women with advanced breast cancer. N Engl J Med 1981;304:16–21.[Abstract]

5 Peethambaram PP, Ingle JN, Suman VJ, Hartmann LC, Loprinzi CL. Randomized trial of diethylstilbestrol vs. tamoxifen in postmenopausal women with metastatic breast cancer. An updated analysis. Breast Cancer Res Treat 1999;54:117–22.[Medline]

6 Shim WS, Conaway M, Masamura S, Yue W, Wang JP, Kmar R, et al. Estradiol hypersensitivity and mitogen-activated protein kinase expression in long-term estrogen deprived human breast cancer cells in vivo. Endocrinology 2000;141:396–405.[Abstract/Free Full Text]

7 Masamura S, Santner SJ, Heitjan DF, Santen RJ. Estrogen deprivation causes estradiol hypersensitivity in human breast cancer cells. J Clin Endocrinol Metab 1995;80:2918–25.[Abstract]

8 Yue W, Santen RJ, Wang JP, Hamilton CJ, Demers LM. Aromatase within the breast. Endocr Relat Cancer 1999;6:157–64.[Abstract/Free Full Text]

9 Jeng MH, Shupnik MA, Bender TP, Westin EH, Bandyopadhyay D, Kumar R, et al. Estrogen receptor expression and function in long-term estrogen-deprived human breast cancer cells. Endocrinology 1998;139:4164–74.[Abstract/Free Full Text]

10 Jeng MH, Yue W, Eischeid A, Wang JP, Santen RJ. Role of MAP kinase in the enhanced cell proliferation of long term estrogen deprived human breast cancer cells. Breast Cancer Res Treat 2000;62:167–75.[Medline]

11 Bacus SS, Gudkov AV, Lowe M, Lyass L, Yung Y, Komarov AP, et al. Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53. Oncogene 2001;20:147–55.[Medline]

12 Evan G, Littlewood T. A matter of life and cell death. Science 1998;281:1317–22.[Abstract/Free Full Text]

13 Lee Y, Renaud RA, Friedrich TC, Gorski J. Estrogen causes cell death of estrogen receptor stably transfected cells via apoptosis. J Steroid Biochem Mol Biol 1998;67:327–32.[Medline]

14 Yao K, Lee ES, Bentrem DJ, England G , Schafer JI, O'Regan RM, et al. Antitumor action of physiological estradiol on tamoxifen-stimulated breast tumors grown in athymic mice. Clin Cancer Res 2000;6:2028–36.[Abstract/Free Full Text]

15 Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science 1998;281:1305–8.[Abstract/Free Full Text]

16 Gutierrez LS, Eliza M, Niven-Fairchild T, Naftolin F, Mor G. The Fas/Fasligand system: a mechanism for immune evasion in human breast carcinomas. Breast Cancer Res Treat 1999;54:245–53.[Medline]

17 Song J, Sapi E, Brown W, Nilsen J, Tartaro K, Kacinski BM, et al. Roles of Fas and Fas ligand during mammary gland remodeling. J Clin Invest 2000;106:1209–20.[Abstract/Free Full Text]

18 Mullauer L, Mosberger I, Grusch M, Rudas M, Chott A. Fas ligand is expressed in normal breast epithelial cells and is frequently up-regulated in breast cancer. J Pathol 2000;190:20–30.[Medline]

19 Mor G, Kohen F, Garcia-Velasco J, Nilsen J, Brown W, Song J, et al. Regulation of fas ligand expression in breast cancer cells by estrogen: functional differences between estradiol and tamoxifen. J Steroid Biochem Mol Biol 2000;73:185–94.[Medline]

20 Sonnenschein C, Szelei J, Nye TL, Soto AM. Control of cell proliferation of human breast MCF7 cells; serum and estrogen resistant variants. Oncol Res 1994;6:373–81.[Medline]

21 Katzenellenbogen BS, Kendra KL, Norman MJ, Berthois Y. Proliferation, hormonal responsiveness, and estrogen receptor content of MCF-7 human breast cancer cells grown in the short-term and long-term absence of estrogens. Cancer Res 1987;47:4355–60.[Abstract]

22 Zhang G, Gurtu V, Kain SR, Yan G. Early detection of apoptosis using a fluorescent conjugate of annexin V. Biotechniques 1997;23:525–31.[Medline]

23 Yoshida A, Shao RG, Pommier Y. Assessment of DNA damage in apoptosis. In: Studzinski GP, editor. Apoptosis. New York (NY): Oxford University Press; 1999. p. 41–237.

24 Kurokawa H, Nishio K, Fukumoto H, Tomonari A, Suzuki T, Saijo N. Alteration of caspase-3 (CPP32/Yama/apopain) in wild-type MCF-7, breast cancer cells. Oncol Rep 1999;6:33–7.[Medline]

25 Diel P, Smolnikar K, Michna H. The pure antiestrogen ICI 182780 is more effective in the induction of apoptosis and down regulation of BCL-2 than tamoxifen in MCF-7 cells. Breast Cancer Res Treat 1999;58:87–97.[Medline]

26 Smolnikar K, Loffek S, Schulz T, Michna H, Diel P. Treatment with the pure antiestrogen faslodex (ICI 182780) induces tumor necrosis factor receptor 1 (TNFR1) expression in MCF-7 breast cancer cells. Breast Cancer Res Treat 2000;63:249–59.[Medline]

27 Koji T, Hishikawa Y, Ando H, Nakanishi Y, Kobayashi N. Expression of Fas and Fas ligand in normal and ischemia-reperfusion testes: involvement of the Fas system in the induction of germ cell apoptosis in the damaged mouse testis. Biol Reprod 2001;64:946–54.[Abstract/Free Full Text]

28 Nilsen J, Mor G, Naftolin F. Estrogen-regulated developmental neuronal apoptosis is determined by estrogen receptor subtype and the Fas/Fas ligand system. J Neurobiol 2000;43:64–78.[Medline]

29 Prendergast GC. Mechanisms of apoptosis by c-Myc. Oncogene 1999;18:2967–87.[Medline]

30 Cobb MH. MAP kinase pathways. Prog Biophys Mol Biol 1999;71:479–500.[Medline]

31 Chen Q, Hung FC, Fromm L, Overbeek PA. Induction of cell cycle entry and cell death in postmitotic lens fiber cells by overexpression of E2F1 or E2F2. Invest Ophthalmol Vis Sci 2000;41:4223–31.[Abstract/Free Full Text]

32 Wang D, Russell JL, Johnson DG. E2F4 and E2F1 have similar proliferative properties but different apoptotic and oncogenic properties in vivo. Mol Cell Biol 2000;20:3417–24.[Abstract/Free Full Text]

33 Hueber AO, Zornig M, Lyon D, Suda T, Nagata S, Evan GI. Requirement for the CD95 receptor-ligand pathway in c-Myc-induced apoptosis. Science 1997;278:1305–9.[Abstract/Free Full Text]

34 Green DR. A Myc-induced apoptosis pathway surfaces. Science 1997;278:1246–7.[Free Full Text]

35 Bissonnette RP, McGahon A, Mahboubi A, Green DR. Functional Myc-Max heterodimer is required for activation-induced apoptosis in T cell hybridomas. J Exp Med 1994;180:2413–8.[Abstract]

36 Yao K, Lee ES, Bentrem DJ, England G, Schafer JI, O'Regan RM, et al. Antitumor action of physiological estradiol on tamoxifen-stimulated breast tumors grown in athymic mice. Clin Cancer Res 2000;6:2028–36.[Abstract/Free Full Text]

37 EL-Ashry D, Miller DL, Kharbanda S, Lippman ME, Kern FG. Constitutive Raf-1 kinase activity in breast cancer cells induces both estrogen-independent growth and apoptosis. Oncogene 1997;15:423–35.[Medline]

38 Suzuki A, Enari M, Eguchi Y, Matsuzawa A, Nagata S, Tsujimoto Y, et al. Involvement of Fas in regression of vaginal epithelia after ovariectomy and during an estrous cycle. EMBO J 1996;15:211–5.[Abstract]

39 Brunner T, Mogil RJ, LaFace D, Yoo NJ, Mahboubi A, Echeverri F, et al. Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas. Nature 1995;373:441–4.[Medline]

40 Joly-Pharaboz MO, Ruffion A, Roch A, Michel-Calemard L, Andre J, Chantepie J, et al. Inhibition of growth and induction of apoptosis by androgens of a variant of LNCaP cell line. J Steroid Biochem Mol Biol 2000;73:237–49.[Medline]

Manuscript received March 26, 2001; revised September 12, 2001; accepted September 18, 2001.


This article has been cited by other articles in HighWire Press-hosted journals:


             
Copyright © 2001 Oxford University Press (unless otherwise stated)
Oxford University Press Privacy Policy and Legal Statement