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[Cancer Research 61, 3902-3907, May 15, 2001]
© 2001 American Association for Cancer Research

Carcinogenesis

Tamoxifen-induced Antitumorigenic/Antiestrogenic Action Synergized by a Selective Aryl Hydrocarbon Receptor Modulator1

Andrew McDougal, Mark Wormke, James Calvin and Stephen Safe2

Departments of Veterinary Physiology and Pharmacology [A. M., M. W., S. S.] and Statistics [J. C.], Texas A&M University, College Station, Texas 77843


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tamoxifen (TAM) is a highly effective selective estrogen receptor (ER) modulator used extensively for the treatment and prevention of breast cancer. However, prolonged treatment of women with TAM may be a risk factor for endometrial cancer, and research in our laboratory is focused on the development of selective aryl hydrocarbon receptor modulators that can be used in combination with TAM to improve its efficacy in the breast and inhibit TAM-induced endometrial effects. This study investigated the effects of the selective aryl hydrocarbon receptor modulators 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF) alone and in combination with TAM in the carcinogen-induced mammary tumor model and in the ovariectomized uterotropic assay using female Sprague Dawley rats. The lowest effective dose of 6-MCDF that inhibited tumor growth was 50 µg/kg/day, and TAM was antitumorigenic at a dose of 100 µg/kg/day. In animals cotreated with TAM + 6-MCDF at doses of 100, 50, or 25 µg/kg/day of each compound, complete inhibition of mammary tumor growth was observed at all doses, and the results are consistent with a more than additive antitumorigenic response for the low dose group (25 + 25 µg/kg) and additive interactions at the 50 and 100 µg/kg doses. In a separate experiment, 6-MCDF (800 µg/kg) inhibited TAM-induced peroxidase activity and progesterone receptor binding in the ovariectomized rat uterus but did not affect TAM-induced bone growth in ovariectomized rats. This study also investigated the effects of TAM and 6-MCDF alone and in combination on ER{alpha} protein levels in MCF-7 human breast cancer cells as a model for studying interactions between these compounds. The results show that 6-MCDF decreased TAM-induced ER{alpha} levels in the absence or presence of 17ß-estradiol through proteasome activation, and these interactions may contribute to the observed combined antitumorigenic effects of these compounds.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breast cancer is among the leading causes of premature death in women, and a high proportion of early stage mammary tumors are ER3 -positive and amenable to endocrine therapy (1) . SERMs, such as the antiestrogen TAM, are used extensively for successful treatment of breast cancer and as chemopreventive agents for women at high risk for this disease (2, 3, 4) . However, there is concern regarding prolonged treatment of women with TAM due to its ER agonist activity in the uterus and the potential increased risk for endometrial cancer (5) . New SERMs such as raloxifene that may pose lower risks for endometrial cancer are being developed as alternative treatments. Additionally, ligands for other receptors including the retinoic acid receptor, peroxisome proliferator-activated receptor, and the vitamin D receptor are also being developed as indirect antiestrogens (6 , 7) because these ligand-activated receptors inhibit estrogen action through complex receptor cross-talk pathways. Because TAM is highly effective for treatment and prevention of breast cancer, it is also important to develop new drugs that can be used in combination with TAM, not only to improve its efficacy in the breast but also to diminish possible adverse side effects, such as endometrial cancer.

Research in this laboratory has focused on AhR-mediated inhibition of E2-induced gene expression and development of SAhRMs for treatment of breast cancer (8) . The AhR was characterized initially by its high binding affinity for the environmental toxicant TCDD (9) , and inhibitory AhR-ER{alpha} cross-talk has been investigated extensively both in vitro and in vivo. In Sprague Dawley rats, TCDD inhibits spontaneous and carcinogen-induced mammary cancers, which are known to be E2 dependent (10 , 11) , and TCDD prevents E2-dependent tumor growth in immunosuppressed B6C3F1 mice bearing MCF-7 breast cancer cell xenografts (12) . In the rodent uterus, TCDD blocks E2-induced hypertrophy, PR and ER binding, peroxidase activity, and expression of the epidermal growth factor receptor and fos genes (11 , 13) . TCDD inhibits E2-induced proliferation, DNA synthesis, and expression of PR and other E2-responsive genes in MCF-7 human breast cancer cells and Ishikawa human endometrial adenocarcinoma cells (13 , 14) . The mechanisms underlying the inhibitory cross-talk between the AhR and ER appear to be complex. TCDD activation of the AhR induces proteasome-mediated degradation of ER{alpha} in breast cancer cells (15) and induces cytochrome P450 enzymes that metabolize E2 and lower cellular levels of this hormone (16) . Another mechanism of inhibitory AhR-ER cross-talk involves direct interactions of the AhR complex with inhibitory dioxin-responsive elements in the 5'-regulatory regions of E2-responsive genes, resulting in inhibition of E2-induced gene expression through multiple gene promoter-specific pathways (17, 18, 19) .

There is evidence that AhR agonists such as TCDD and polynuclear aromatic hydrocarbons inhibit mammary and endometrial tumor formation in humans (8 , 20 , 21) . Our laboratory has developed two classes of relatively nontoxic AhR agonists, namely, alternate substituted alkyl polychlorinated dibenzofurans and diindolylmethane analogues, that inhibit growth of DMBA-induced mammary tumor in female Sprague Dawley rats (22, 23, 24, 25) ; moreover, these compounds also exhibit antiestrogenic activity in the rodent uterus (8 , 23 , 25 , 26) . Both classes of SAhRMs have minimal effects on classical AhR-mediated toxic responses observed in most rodent and cell culture models. Moreover, 6-MCDF, the prototypical SAhRM used in this study, inhibits TCDD-induced CYP1A1 gene expression, immunotoxicity, porphyria, and teratogenicity in mice through partial AhR antagonism (8) .

This study describes inhibition of carcinogen-induced mammary tumor growth in female Sprague Dawley rats by TAM, 6-MCDF, and their combination and antagonism of TAM-induced uterine effects by 6-MCDF in the ovariectomized rat model, suggesting that both drugs in combination may be highly effective for treating breast cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Maintenance and Western Blot Analysis.
MCF-7 human breast cancer cells were obtained from American Type Culture Collection (Manassas, VA). Cells were grown on monolayer cultures in DMEM:Ham’s F-12 media with phenol red supplemented with 2.2 grams/liter sodium bicarbonate, 0.2 gram/liter BSA, 10 mg/liter apo-transferrin, 5% fetal bovine serum (Intergen, Purchase, NY), and antibiotic-antimycotic solution (pH 7.4). Cells were seeded into 35-mm 6-well plates in phenol-free media containing 2.5% charcoal-stripped fetal bovine serum. The next day, cells were treated in fresh media for 24 h with the following regimens: (a) DMSO as a control vehicle (0.1% v/v); (b) 1 nM E2; (c) 1 µM 6-MCDF; (d) 1 nM E2 + 1 µM 6-MCDF; (e) 1 µM 4-OHTAM; (f) 1 µM 4-OHTAM + 1 µM 6-MCDF; (g) 1 µM 4-OHTAM + 1 nM E2; and (h) 1 µM 4-OHTAM + 1 nM E2 + 1 µM 6-MCDF. Cells were washed once with PBS and collected by scraping in 200 µl of lysis buffer [50 mM HEPES, 0.5 M NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% (v/v) glycerol, 1% Triton X-100, 10 µg/ml aprotinin, 50 mM phenylmethylsulfonyl fluoride, and 50 mM sodium orthovanadate]. The lysates were incubated on ice for 1 h with intermittent vortexing followed by centrifugation (40,000 x g, 10 min, 4°C). Equal amounts of protein from each treatment group, as well as pure Sp1 protein (Promega Corp., Madison, WI) and in vitro translated ER{alpha} (15) standards, were separated by SDS-PAGE in a 7.5% acrylamide/bisacrylamide gel and electrophoresed to polyvinylidene difluoride membrane in transfer buffer (48 mM Tris, 39 mM glycine, and 0.025% SDS). Membranes were blocked for 15 min in Blotto (5% milk + TBS) and then probed with the polyclonal antibodies for ER{alpha} (sc-544; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:1,000 dilution in Blotto or with antibodies for Sp1 at a 1:10,000 dilution (sc-59; Santa Cruz Biotechnology, Inc.). After 5 h, the membrane was washed twice (5 min each time) in TBS + 0.05% Tween 20, probed with secondary peroxidase-conjugated antibody (diluted 1:5,000 in Blotto) for 2 h, washed three times (5 min each time) in TBS + 0.05% Tween 20, washed once for 5 min in TBS, and then visualized using the enhanced chemiluminescence detection system (New England Nuclear, Boston, MA). ER{alpha} protein levels in each treatment group were determined relative to the levels in the vehicle control (DMSO) group, which were set at 100%. Results are expressed as the means + SE for at least three separate determinations for each treatment group. The Western blot analysis of Sp1 protein was derived from a single experiment.

Mammary Tumor Studies.
Virgin female Sprague Dawley rats were obtained from Harlan (Houston, TX) and treated p.o. at 50 days of age with a single dose of DMBA (20 mg/rat). Within 45–75 days, tumors could be detected by palpation; when the tumors reached a small predetermined size (100–200 mm3), rats were treated by gavage with corn oil (vehicle) or test compounds dissolved in corn oil daily for 20 days at a final volume of 2 ml/kg, with 8–10 rats/treatment group (22, 23, 24) . The assay was normally limited to 1 tumor/animal; in a few cases where two tumors of equal size were observed in the same animal, the daily tumor volumes were averaged to give a single data point per animal. Tumors were measured with calipers, and volume was calculated by the formula: (length/2) x (width/2) x (length/2) x {pi}. Rats were euthanized on day 21. Tumors and selected organs were excised, weighed, and processed for histopathological examination. Hepatic microsomal extracts were prepared and analyzed for EROD activity as described previously (22, 23, 24) .

Uterine and Bone Study in Ovariectomized Rats.
Cycling female virgin Sprague Dawley rats were ovariectomized at approximately 90 days of age. Animals were allowed to acclimate for 3 weeks before treatments to allow serum hormone levels to normalize and so that the age of the ovariectomized rats would correspond to the age of rats in the tumor studies. Rats were treated by gavage with corn oil (vehicle) or test compounds in corn oil daily for 20 days; four rats received control vehicle; five rats were treated with 400 µg/kg TAM, five rats were treated with 800 µg/kg 6-MCDF; and six rats were cotreated with 400 µg/kg TAM + 800 µg/kg 6-MCDF. On day 21, animals were euthanized, and uteri were immediately excised, weighed, bisected, and processed; livers were weighed and frozen in liquid nitrogen so that hepatic microsomes could be prepared and tested for EROD activity, and other organs (heart, spleen, and kidneys) were weighed and checked for gross pathology. The uterine cytosolic progesterone receptor assay, calculated as fmol receptor/uterus, and the uterine nuclear peroxidase activity assay, calculated as absorbance units/min/uterus, were performed as described previously (23) . Uterine wet weight was measured after blotting and before bisecting each uterus. Left and right femurs were excised, and their length was measured with calipers; the femurs were then washed for 4 h with PBS (pH 2.0) to soften muscle and connective tissue, allowing bones to be completely cleaned before blot drying and measurement of wet weight. Femurs were dried for 12 h at 130°C and allowed to cool, and dry weight was measured. Data were normalized to body weight and are expressed as a percentage of control.

Data Analysis.
Statistical differences among groups were determined using ANOVA and Duncan’s new multiple range. Results are expressed as the means ± SE. The fractional method of Webb (27 , 28) and the simple isobologram method of Lowe (27 , 29) were used to determine synergistic interactions of 6-MCDF and TAM. The Q test for removal of outliers was performed for the interaction studies, and one rat from the control group, one rat from the TAM (50 µg/kg)-treated group, and one rat from the 6-MCDF + TAM (50 µg/kg)-treated group were removed.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of Proteasome Pathways by 6-MCDF.
Fig. 1Citation shows the separate and combined effects of a 24-h treatment with E2, 6-MCDF, and 4-OHTAM on intracellular ER{alpha} and Sp1 protein levels in MCF-7 human breast cancer cells. E2 and 6-MCDF alone each decreased ER{alpha} levels relative to control, and treatment with E2 + 6-MCDF caused a further decrease in ER{alpha} levels. Treatment with proteasome inhibitors blocked ER down-regulation by E2, 6-MCDF, and AhR agonists (data not shown; Ref. 15 ). 4-OHTAM, the active metabolite of TAM, significantly increased ER{alpha} levels and prevented the E2-induced degradation of ER and cotreatment with 6-MCDF partially reversed this effect of 4-OHTAM, in both the presence and absence of E2. Levels of Sp1, a transcription factor important in basal expression of many different genes, were not affected by treatment and serve as a control for this study as described previously (15) .



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  		Fig. 1. ER{alpha} protein levels are modulated through the ER and AhR pathways. Western blot analysis was performed on 30 µg of protein from each treatment group separated by SDS PAGE, and the resulting autoradiograph was quantitated using a densitometer. C, control treatment; E, treatment with 1 nM E2; M, treatment with 1 µM 6-MCDF; and 4T, treatment with 4-OHTAM. STD, the loading of pure Sp1 protein and in vitro translated ER{alpha} as standards. E2 and 6-MCDF treatment significantly (P < 0.05) decreased ER{alpha} levels compared with control treatment, and treatment with E2 + 6-MCDF resulted in a further decrease in ER{alpha} levels. 4-OHTAM treatment significantly (P < 0.05) increased ER{alpha} levels compared with controls; ER{alpha} down-regulation by E2 was blocked after cotreatment with 4-OHTAM. 6-MCDF significantly (P < 0.05) decreased induction of ER{alpha} protein by 4-OHTAM in the presence or absence of E2. Results are the means ± SEs for at least three separate experiments for each treatment group, and representative Western blots are shown. Means with P < 0.05 less than controls are indicated with an a, means with P < 0.05 less than E2 treatment are indicated with an ab, and means with P < 0.05 less than 4-OHTAM treatment are indicated with a c.

 
Interaction of TAM and 6-MCDF on Inhibition of Mammary Tumor Growth.
The effects of TAM, 6-MCDF, or TAM + 6-MCDF on the growth of DMBA-induced mammary tumors (adenocarcinomas) in Sprague Dawley rats are summarized in Fig. 2Citation and Table 1Citation . The lowest effective doses of TAM and 6-MCDF that inhibited mammary tumor growth were 100 and 50 µg/kg/day, respectively, whereas doses of 50 and 25 µg/kg/day (TAM) or 25 µg/kg/day (6-MCDF) were inactive. In contrast, combined treatment with TAM + 6-MCDF using doses of 100, 50, or 25 µg/kg/day of each compound resulted in virtually complete inhibition of mammary tumor growth, and in some of the treatment groups, final tumor volumes were less than the initial volumes, indicating some growth stasis. Interactions were tested by comparing tumor volumes and by comparing the rate of tumor growth (slope of linear least squares fit). The fractional product method of Webb (27 , 28) showed that the interactions of TAM + 6-MCDF at doses of 100 and 50 µg/kg are additive, and the interaction at 25 µg/kg is synergistic. This method was designed to compare the fractional inhibition of a response by two inhibitors with their effect in combination. The simplified isobologram method of Lowe (27 , 29) distinguished a synergistic interaction at 25 µg/kg by comparing growth rates but was not robust enough to distinguish synergism from additivity for the interactions at 100 and 50 µg/kg. This analysis compares the effect of the addition of a second drug to the activity of the first drug against the effect of the addition of more of the first drug to itself. Body and organ (uterus, liver, heart, spleen, and kidneys) weights were unchanged in all treatment groups. No induction of hepatic CYP1A1-dependent EROD activity was observed in animals treated with 6-MCDF (alone or in combination), and this was consistent with previous studies showing that 6-MCDF induced EROD activity in rodents only at doses > 50 mg/kg (30) .



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  		Fig. 2. Inhibition of DMBA-induced rat mammary tumor growth by TAM and 6-MCDF. Rats were treated by gavage with corn oil (vehicle) or test compounds dissolved in corn oil daily for 20 days and euthanized on day 21. The study is presented in three panels as tumor volume versus time in days, and the corn oil control data are shown for each study. Control tumors weighed 16.0 ± 6 g. A, effects of TAM treatment. Treatment groups were control and TAM (100, 50, or 25 µg/kg). B, effects of 6-MCDF treatment. Treatment groups were control and 6-MCDF (100, 50, or 25 µg/kg). C, effects of cotreatment with TAM + 6-MCDF. Treatment groups are control and TAM (100 µg/kg) + 6-MCDF (100 µg/kg), TAM (50 µg/kg) + 6-MCDF (50 µg/kg), or TAM (25 µg/kg) + 6-MCDF (25 µg/kg). *, significant differences (P < 0.05) in tumor weights and volumes compared with those observed for control-treated animals. Bars, SE.

 

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  		Table 1 Effects of TAM, 6-MCDF, and their combinations on final body weight, tumor volumes and weights in rats bearing mammary tumorsa

 
Interaction of TAM and 6-MCDF on Uterine Responses and Bone Growth.
The ovariectomized rat is a classical model for measuring the effects of SERMs such as TAM in uterus and bone (31) . Interactions of TAM + 6-MCDF were determined in ovariectomized female Sprague Dawley rats using an estrogenic dose of TAM (400 µg/kg/day) for 20 days in combination with 6-MCDF at 800 µg/kg/day (Fig. 3Citation and Table 2Citation ) or 400 µg/kg (data not shown). TAM significantly induced uterine wet weight, peroxidase activity, and levels of progesterone receptor binding. 6-MCDF (800 µg/kg/day) significantly inhibited uterine peroxidase activity and did not induce wet weight or PR binding. In animals treated with TAM + 6-MCDF, minimal changes in uterine wet weight were observed compared with treatment with TAM alone; however, TAM + 6-MCDF (400 and 800 µg/kg/day) significantly inhibited uterine peroxidase activity, and TAM + 6-MCDF (800 µg/kg) inhibited PR binding (Fig. 3ACitation and Table 2Citation ). These results show that 6-MCDF inhibits some TAM-induced uterine responses, and this is consistent with a previous study showing that a single dose of 43 mg/kg 6-MCDF completely inhibited E2-induced uterine weight increase and PR induction in the immature rat (26) . In the same ovariectomized female Sprague Dawley rats, treatment with TAM alone (400 µg/kg/day) resulted in significantly increased femur length and wet and dry weight. 6-MCDF (800 µg/kg/day) alone had no effect on bone growth, and the effects of treatment with TAM + 6-MCDF did not differ from those observed for treatment with TAM alone (Fig. 3BCitation and Table 2Citation ). ER agonists inhibit weight gain in ovariectomized adult rats (31 , 32) , and in this study, TAM (with or without 6-MCDF) decreased rat body weight over the 21-day study (7% and 3%, respectively; Table 2Citation ). 6-MCDF had no effect on body weight compared with controls, and significant weight gain was observed in the two groups of rats over the 21-day study (12% and 16%, respectively; Table 2Citation ).



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  		Fig. 3. Inhibition of TAM-induced estrogenic effects in ovariectomized rats. Rats were treated by gavage with corn oil (vehicle) or test compounds in corn oil daily for 20 days and euthanized on day 21. No changes in organ weight (as a percentage of body weight), gross pathology, or hepatic EROD activity were detected. Means are expressed as a percentage of control ± SE. Statistical analysis used ANOVA and Duncan’s new multiple range (a two-tailed test); means with P < 0.05 from controls are indicated by an a and means with P < 0.05 from TAM-treated animals are indicated by a b. A, uterine responses. Uterine cytosolic progesterone receptor binding and uterine nuclear peroxidase activity are calculated as fmol/uterus, and absorbance is calculated as units/min/uterus, and they are shown as a percentage of control values. Uterine wet weight was measured after blotting and before bisecting each uterus, normalized to body weight, and also expressed as a percentage of control. Numerical values are shown in Table 2Citation . B, bone responses. Left and right femurs were excised and weighed, and their length was measured with calipers. Femurs were dried for 12 h at 130°C and allowed to cool, and dry weight was measured (31) . Data were normalized to body weight and expressed as a percentage of control (Table 2)Citation .

 

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  		Table 2 Effects of TAM, 6-MCDF, and their combination on body weight (BW) and organ weights and uterine and bone responses in ovariectomized ratsa

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The remarkable efficacy of the combined therapy (TAM + 6-MCDF) is evident from the low-dose treatment (25 µg/kg/day) in which the compounds alone were inactive, but the compounds in combination inhibited mammary tumor growth. Although it is difficult to definitively characterize drug interactions in small studies, the application of two different test models (27, 28, 29) to our data indicates that the antitumorigenic effects of 6-MCDF and TAM at the 25 + 25 µg/kg dose level were synergistic and are additive at the 50 + 50 and 100 + 100 µg/kg doses for inhibition of rat mammary tumor growth. Additionally, 6-MCDF inhibited two TAM-induced markers of estrogenicity in the uterus without affecting the desirable ER agonist activity of TAM on bone growth (Fig. 3BCitation and Table 2Citation ) or body weight, and this is consistent with other studies indicating that 6-MCDF blocks the uterotropic effects of estrogen in a dose-dependent fashion (8 , 26) . These results demonstrate that SAhRMs acting through inhibitory AhR-ER cross-talk pathways are not only potent antitumor agents but also markedly enhance the effects of TAM as an antiestrogen in the carcinogen-induced rat mammary tumor model. Thus, the use of 6-MCDF and other SAhRMs in combination with direct-acting antiestrogens such as TAM may lower the effective dose of both compounds for treatment of human breast cancer while affording protection from SERM-induced estrogenic responses in the uterus.

Both AhR agonists and TAM inhibit the growth of DMBA-induced mammary tumors in rats (Fig. 2)Citation and tumors in mice bearing MCF-7 cell xenografts (12 , 25) . In vitro, 6-MCDF and TAM alone also inhibit E2-induced growth of MCF-7 cells and expression of E2-regulated genes through inhibitory AhR-ER{alpha} cross-talk (for 6-MCDF; Ref. 8 ) and direct interactions with ER{alpha} (for TAM; Ref. 2 ). Mechanisms of action of 6-MCDF and TAM are complex and are likely to be gene specific (2) . Recent studies showed that treatment of MCF-7 cells with E2 resulted in proteasome-dependent degradation of ER{alpha}, and this response modulates the duration of hormone-induced activation of transcription (15 , 33, 34, 35) . Our laboratory has also shown that AhR agonists such as TCDD and 6-MCDF simultaneously induce proteasome-dependent degradation of the AhR and ER{alpha} in MCF-7 and T47D breast cancer cells (15) . Moreover, in MCF-7 cells treated with E2 + TCDD or 6-MCDF, dual activation of proteasome pathways resulted in even lower levels of immunoreactive ER{alpha}, and this may contribute to inhibitory AhR-ER{alpha} cross-talk by limiting cellular levels of ER{alpha}.

Using MCF-7 cells as a model, we have investigated the effects of 4-OHTAM, 6-MCDF, and their combination on ER{alpha} levels. Because the growth of DMBA-induced mammary tumors and MCF-7 xenografts in rodent models is dependent on E2, the effects of 4-OHTAM and 6-MCDF in combination with E2 were also investigated. Fig. 1Citation shows that TAM, alone and in combination with E2, increases levels of ER{alpha} protein in whole cell extracts, and this contrasts with the down-regulation of ER{alpha} observed after treatment with E2 alone, 6-MCDF alone, or E2 + 6-MCDF. These responses are blocked by proteasome inhibitors (Ref. 15 ; data not shown). It is paradoxical that 4-OHTAM increases ER{alpha} protein in MCF-7 cells (Fig. 1)Citation but inhibits cell proliferation, and this has been observed previously in breast cancer cells (36, 37, 38) . In contrast, TAM/4-OHTAM, which acts as an estrogen in the endometrium, decreases ER levels in this tissue (39, 40, 41, 42) . Cell context-dependent effects of TAM on ER{alpha} may be related to altered subcellular distribution or phosphorylation of ER{alpha}, and this is currently being investigated. Additionally, it has been suggested that the conformational changes induced in ER by TAM that modulate coactivator binding and transcriptional activation may also affect recruitment of proteins necessary for proteasome-mediated degradation of ER{alpha} (36 , 43) . However, our results show that the combined effects of 6-MCDF + 4-OHTAM alone and in combination with E2 result in significantly decreased ER{alpha} protein levels compared with those observed after treatment with 4-OHTAM alone. Additional studies are required to elucidate the degree to which TAM-induced ER stabilization or destabilization induces tissue-specific estrogenic and antiestrogenic responses and how the degradation of ER{alpha} in the presence of SAhRMs modulates these effects. These interactions may play a significant role in mediating the potent combined effects of 6-MCDF and TAM as inhibitors of mammary tumor growth in the DMBA-induced rat mammary tumor model.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the NIH Grants CA-64801 and ES09160, AVAX Technologies (Kansas City, MO), and the Texas Agricultural Experiment Station. S. S. is a Sid Kyle Professor of Toxicology. Back

2 To whom requests for reprints should be addressed, at Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843-4466. Phone: (979) 845-5988; Fax: (979) 862-4929; E-mail: ssafe{at}cvm.tamu.edu Back

3 The abbreviations used are: ER, estrogen receptor; SERM, selective ER modulators; TAM, tamoxifen; AhR, aryl hydrocarbon receptor; SAhRM, selective AhR modulators; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; PR, protein receptor; DMBA, 7,12-dimethylbenz(a)anthracene; 6-MCDF, 6-methyl-1,3,8-trichlorodibenzofuran; 4-OHTAM, 4-hydroxytamoxifen; E2, 17ß-estradiol; TBS, Tris-buffered saline [10 mM Tris and 150 mM NaCl (pH 8.0)]; EROD, ethoxyresorufin O-deethylase. Back

Received 10/16/00. Accepted 3/15/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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