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).
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ABSTRACT |
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INTRODUCTION |
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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 35 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-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.
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MATERIALS AND METHODS |
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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-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, UrbanaChampaign, 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. 23375212; 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 13 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.
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RESULTS |
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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. 1). 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. 1
). 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.
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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, A, 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, A
).
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Agarose gel electrophoresis was used to confirm the DNA fragmentation as further evidence of estradiol-induced apoptosis in the LTED cells (Fig. 2, C). 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, D). 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, D
). 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, D
). 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. 3). 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.
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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, A). 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, B
) and in MCF-7 cells (data not shown). Expression of Fas protein in LTED cells was not affected by estradiol treatment.
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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 panel). Furthermore, a combination of anti-Fas antibody with estradiol had an additive effect on apoptosis of LTED cells (Fig. 5, A; left panel
), 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 panel
). 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 panel
).
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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, A, 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, B
). It is interesting that BSK3 cells did not respond to estradiol by either inducing apoptosis or altering cell growth (Fig. 6, A and B
). Further studies revealed that the basal level of Fas protein in E8CASS cells was increased, whereas that of BSK3 was not (Fig. 6, C
).
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DISCUSSION |
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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 (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
undergo apoptosis, whereas cells with ER
are protected from apoptosis (28). Although the mechanisms underlying these effects of estradiol are not clear, apoptosis mediated by ER
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 doseresponse 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.
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NOTES |
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Manuscript received March 26, 2001; revised September 12, 2001; accepted September 18, 2001.
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