help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stoica, A.
Right arrow Articles by Martin, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stoica, A.
Right arrow Articles by Martin, M. B.
Endocrinology Vol. 141, No. 10 3595-3602
Copyright © 2000 by The Endocrine Society

ARTICLES

Effects of Arsenite on Estrogen Receptor-{alpha} Expression and Activity in MCF-7 Breast Cancer Cells1

Adriana Stoica, Elizabeth Pentecost and Mary Beth Martin

Department of Biochemistry and Molecular Biology, Lombardi Cancer Center, Georgetown University, Washington, D.C. 20007

Address all correspondence and requests for reprints to: Dr. Mary Beth Martin, Lombardi Cancer Center, E411 Research Building, 3970 Reservoir Road NW, Washington, D.C. 20007. E-mail: martinmb{at}gunet.georgetown.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine whether arsenite has estrogen-like activities, the effects of this compound on estrogen receptor-{alpha} (ER{alpha}) and other estrogen-regulated genes were measured in the human breast cancer cell line MCF-7. Treatment of cells with 1 µM arsenite resulted in a 60% decrease in the amount of ER{alpha} and in a parallel decrease of 40% in ER{alpha} messenger RNA. Progesterone receptor concentration increased 22-fold after arsenite treatment. pS2 messenger RNA also increased 2.1-fold after treatment. The induction of progesterone receptor and pS2 was blocked by the antiestrogen ICI-182,780. In transient cotransfection experiments of wild-type ER{alpha} and an estrogen response element-reporter construct, arsenite stimulated chloramphenicol acetyltransferase (CAT) activity. In growth assays, arsenite significantly stimulated the proliferation of MCF-7 cells compared with cells grown in estrogen-depleted medium. Addition of an antiestrogen blocked growth stimulation by arsenite. In binding assays, arsenite blocked the binding of estradiol to ER{alpha} (Ki = 5 ± 0.5 nM; n = 3), suggesting that the compound interacts with the hormone-binding domain of the receptor. To determine whether interaction of arsenite with the hormone-binding domain results in receptor activation, COS-1 cells were transiently cotransfected with the chimeric receptors GAL-ER, which contains the hormone-binding domain of ER{alpha} and the DNA-binding domain of the transcription factor GAL4, and a GAL4-responsive CAT reporter gene. Treatment of cells with estradiol or arsenite resulted in a 4-fold increase in CAT activity. The effects of arsenite on the chimeric receptor were blocked by the antiestrogen, suggesting that arsenite activates ER{alpha} through an interaction with the hormone-binding domain of the receptor. Transfection assays with ER{alpha} mutants identified C381, C447, H524, and N532 as interaction sites of arsenite with the hormone-binding domain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BREAST CANCER IS the most common malignancy affecting women and is the leading cause of death in women between the ages of 35–45 yr (1). Epidemiological studies suggest that endocrine factors play a pivotal role in the etiology of the disease (1). The primacy of hormonal factors in the etiology of breast cancer reflects the control of proliferation by estrogens. Because estrogen receptor-{alpha} (ER{alpha}) is a critical mediator of growth, molecules that can bind to and activate ER{alpha} can potentially increase the risk for breast cancer. A number of natural and man-made chemicals have been identified in the environment that possess estrogenic activity and, therefore, may pose a health risk. This study provides evidence that arsenite is a new candidate environmental estrogen.

Arsenic is an element that has no known physiological function but is present in the body as a result of environmental exposure (2, 3). It is widely distributed in the environment; the primary source of human exposure is through drinking water and food (4). In addition to diet, medical and occupational exposures are important sources of both inorganic and organic arsenic. Most occupational exposures occur in smelters (5, 6); during the manufacture of insecticides, pesticides, fungicides, and pharmaceutical substances; and during the production of wines. Inorganic arsenic is converted in the liver to various oxidation states, including arsenite, arsenate, and arsenide, and subsequently is methylated to various organometalloidal forms, such as dimethylarsinic acid. The methylated species are excreted in the urine, but may be deposited in lungs, liver, kidney, hair, and nails (4). Cohort studies in humans have demonstrated an association between long-term exposure to arsenic and lung and urinary cancers as well as an association with bladder, kidney, and liver cancers (7, 8, 9, 10). Further, a possible link between arsenic in drinking water and the prevalence of skin and prostate cancer has been demonstrated (11, 12, 13, 14, 15). Although arsenic is a well documented carcinogen in humans, evidence for its carcinogenicity in animals is limited (16, 17). In mice, arsenic is inactive as either an initiator or a promoter of tumorigenesis (18); however, exposure to dimethylarsinic acid strongly promotes lung tumors after initiation with 4-nitroquinoline-1-oxide (19). In cells in culture, arsenic induces morphological transformation (20, 21), increases the frequency of sister chromatid exchange (22), and causes chromosomal alterations and gene amplification (4, 18, 22), but it fails to induce mutations at specific loci (21). Although the precise mechanism by which arsenic induces DNA damage is not known, the compound alters the activity of enzymes involved in DNA repair and interferes with DNA synthesis through the interaction of the arsenite ion (As3+) with thiol groups (17), suggesting that arsenic is a potentiator, rather than a direct carcinogen, in tumorigenesis.

Previous studies from this laboratory have demonstrated that the heavy metal cadmium is a candidate environmental estrogen. The metal mimics the effects of estradiol in estrogen-responsive breast cancer cell lines (23) through a high affinity interaction with the hormone-binding domain of ER{alpha} (24). Cadmium also interacts with the hormone-binding domain of the glucocorticoid receptor and blocks dexamethasone binding (25); however, cadmium does not activate the glucocorticoid receptor (24). Arsenite has also been shown to bind to the hormone-binding domain of the glucocorticoid receptor and block dexamethasone binding (25). To determine whether arsenite is a potential environmental estrogen, the effects of arsenite on ER{alpha} expression and activity in the ER-positive breast cancer cell line MCF-7 were studied. Arsenite induced the estrogen-regulated genes progesterone receptor and pS2 and increased the growth of the cells. The compound appears to activate ER{alpha} through an interaction with the hormone-binding domain that also blocked estradiol binding to the receptor. The interaction of arsenite with ER{alpha} involves several amino acids in the hormone-binding domain, suggesting that the compound may form a coordination complex within the hormone-binding domain and thereby activate ER{alpha}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue culture
MCF-7 human breast cancer cells were grown in improved MEM (IMEM) supplemented with 5% FCS. At 70% confluence, the medium was changed to phenol red-free IMEM supplemented with 5% charcoal-stripped calf serum. Calf serum was pretreated with dextran-coated charcoal to remove endogenous steroids. Cells were maintained in this medium for 2 days before treatment and were then treated with sodium arsenite, estradiol (Sigma, St. Louis, MO), or the steroidal antiestrogen ICI-182,780 (Zeneca Pharmaceuticals, Wilmington, DE).

Measurement of ER{alpha} and progesterone receptor protein concentration
Cells were grown as described above. After 24-h treatment with sodium arsenite, the cells were washed twice with PBS and pelleted by centrifugation. Cell pellets were sonicated in a high salt buffer (26), and the homogenate was incubated on ice for 30 min and centrifuged at 100,000 x g for 1 h at 4 C. Supernatants were assayed for ER{alpha} and progesterone receptor protein. The concentrations of ER{alpha} and progesterone receptor protein were determined using specific enzyme immunoassay kits from Abbott Laboratories (North Chicago, IL). Aliquots of the total extracts were analyzed according to the manufacturer’s instructions.

Measurement of ER{alpha} and pS2 mRNA amounts
Total cellular RNA was extracted from cells as described previously (26). The amounts of ER{alpha} and pS2 mRNA were determined by a ribonuclease (RNase) protection assay. 32P-Labeled antisense RNA (cRNA) was synthesized in vitro from pOR300 (estrogen receptor) (26), 36B4 (26), and pS2 (27) using T7 polymerase. Sixty micrograms of total RNA were hybridized for 16 h to the 32P-labeled cRNAs. The protected cRNA probes were resolved on 6% polyacrylamide gels. The bands were visualized by autoradiography and quantified by phosphorimaging. The amounts of ER{alpha} and pS2 mRNA were normalized using 36B4 as an internal control.

Transient transfection assays
A low temperature and low pH calcium phosphate method was employed to transfect COS-1 cells (28). COS-1 cells were plated at a density of 3 x 106 cells/150-mm dish in phenol red-free IMEM containing 10% charcoal-stripped calf serum for 24 h. The cells were transfected with 120 µg DNA containing 15 µg of an ER{alpha} expression vector (wild type or mutant, as described below), 75 µg of the reporter construct pb-CAT (S)MERE, 6 µg ß-galactosidase, and salmon sperm carrier DNA. Sixteen to 18 h after transfection, the precipitate was washed off, and the cells were replenished with phenol red-free IMEM containing 10% charcoal-stripped serum in the presence or absence of 1 nM estradiol or 1 µM sodium arsenite. The cells were harvested 24 h later, and chloramphenicol acetyltransferase (CAT) activity was measured as described previously (23). CAT activity was expressed as the percent conversion of chloramphenicol to its acetylated forms and was normalized to the activity of ß-galactosidase. The increase in CAT activity in response to treatment was expressed relative to that in untreated controls. Expression vectors for the wild-type ER{alpha} and the amino acid mutants (C381A, C417A, C447A, C530A, E523A, D538N, H524A, K529Q, K531Q, and N532D) were described previously (29, 30, 31, 32). For these transient transfection assays, the estrogen-responsive reporter construct pbCAT-(S)MERE (33) was obtained from Dr. D. El Ashry (Lombardi Cancer Center, Georgetown University, Washington DC). The chimeric receptors GAL-ER and GAL-GR and the reporter plasmid 17 M2GCAT were also described (34).

ER{alpha} binding assays
The ability of arsenite to block estradiol binding to ER{alpha} was determined in cell extracts from MCF-7 cells that were maintained in phenol red-free IMEM containing 5% charcoal-stripped serum. After 2 days in estrogen-depleted medium, the cells were lysed by sonication in a high salt buffer containing 10 mM Tris (pH 7.5), 1.5 mM EDTA, 5 mM sodium molybdate, 0.4 M KCl, 1 mM monothioglycerol, and 2 mM leupeptin. The homogenate was incubated on ice for 30 min and centrifuged at 100,000 x g for 1 h at 4 C (35). The protein concentration of the cell extract was determined by the Bradford method. Cell extracts were preincubated on ice with various concentrations of sodium arsenite (1 pM to 10 µM). [3H]Estradiol (10 nM) was then added in the presence and absence of a 200-fold molar excess of diethylstilbestrol and incubated at 4 C for 16–18 h. Free steroid was removed by the addition of 5% dextran-coated charcoal. The amount of radioactivity was measured by scintillation counting. Specifically bound complexes were calculated by subtracting nonspecific binding from total binding.

Anchorage-dependent growth assays
MCF-7 cells were plated at 105 cells/well into six-well plates in IMEM supplemented with 5% FCS. Cells were grown to 40% confluence, and the medium was changed to phenol red-free IMEM supplemented with 5% charcoal-stripped serum. After 2 days in this medium, cells were treated with either 1 nM estradiol or 1 µM sodium arsenite. Medium with the appropriate treatments was replaced every 2 days. Cells were trypsinized at the specific time points and counted with a Coulter counter (Coulter Electronics, Inc., Hialeah, FL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of arsenite treatment on the concentration of ER{alpha} protein
To determine the effect of arsenite on the concentration of ER{alpha} protein, an enzyme immunoassay was employed. MCF-7 cells were treated with several concentrations of arsenite (0.1–5 µM) for 24 h (Fig. 1Go). The effect of arsenite was concentration dependent. The ER{alpha} concentration decreased from 453 fmol/mg protein in control cells to 227, 181, and 45 fmol/mg protein (50%, 60%, and 90% decrease) after 0.1, 1, and 5 µM arsenite treatment, respectively.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. Effect of arsenite on the concentration of ER{alpha}. MCF-7 cells were grown in IMEM supplemented with 5% FCS. At 80% confluence, medium was changed to phenol red-free IMEM and 5% charcoal-treated calf serum. Cells were grown in this medium for 2 days and then treated for 24 h with 0.1, 1, or 5 µM sodium arsenite or 1 nM estradiol. ER{alpha} protein was determined using an enzyme immunoassay as described in Materials and Methods and was expressed as a percentage of that in control cells (mean ± SD; n = 5).

 
Effect of arsenite on the steady state amount of ER{alpha} messenger RNA (mRNA)
To determine whether the reduction in ER{alpha} protein paralleled a reduction in the steady state amount of ER{alpha} mRNA, a RNase protection assay was performed. MCF-7 cells were treated with 0.1, 1, or 5 µM arsenite, and the effects of treatment on the steady state amount of total ER{alpha} mRNA were measured. In these experiments the amount of ER{alpha} mRNA was quantified by phosphorimaging and normalized to the amount of 36B4 mRNA. The 36B4 transcript encodes for the ribosomal acidic protein PO (36), which is constitutively expressed in the presence of estradiol and growth factors (26, 27, 35, 37). The data are presented in Fig. 2Go as a percentage of the control value for the ratio of the ER{alpha} signal to the 36B4 signal. In this study treatment with 1 nM estradiol resulted in a 70% decrease in ER{alpha} mRNA amounts, which is in agreement with our previous observations (26). Arsenite treatment of MCF-7 cells resulted in an approximately 40% decrease in ER{alpha} mRNA in cells treated with either 0.1 or 1 µM. Treatment with 5 µM arsenite resulted in an approximately 90% decrease in receptor mRNA. The decrease in ER{alpha} mRNA after treatment with arsenite roughly correlated with the magnitude of the effect on ER{alpha} protein amounts.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 2. Effect of arsenite on the steady state amount of ER{alpha} mRNA. MCF-7 cells were grown as described in Fig. 1Go and were treated for 24 h with 0.1, 1, or 5 µM sodium arsenite or 1 nM estradiol. Total cellular RNA was extracted, and ER{alpha} mRNA was determined by a RNase protection assay as described in Materials and Methods. Autoradiographs were quantified by phosphorimaging, and the values were represented as the ratio of the ER{alpha} signal to 36B4 signal. Results are presented as a percentage of the control values and represent the mean value of six experiments ± SD.

 
Effect of arsenite treatment on the concentration of progesterone receptor protein
To determine the effect of arsenite on the amount of progesterone receptor, an enzyme immunoassay was performed. MCF-7 cells were treated with 1 µM arsenite for 24 h, and the concentration of progesterone receptor was measured (Fig. 3Go). In response to treatment with arsenite, the progesterone receptor concentration increased 22-fold compared with control levels. The magnitude of this increase was similar to the increase in progesterone receptor concentration after treatment with 1 nM estradiol. Treatment with 1 nM estradiol resulted in a 14-fold increase in progesterone receptor over control values. To determine whether the effects of arsenite were mediated by ER{alpha}, the ability of the antiestrogen ICI-182,780 (500 nM) to block the effect of arsenite was tested. As expected, the antiestrogen had no effect on progesterone receptor expression and blocked induction by estradiol. The antiestrogen also blocked the effects of arsenite, suggesting that the effects of the compound are mediated by ER{alpha}.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 3. Effect of arsenite on the progesterone receptor protein. MCF-7 cells were grown as described in Fig. 1Go and were treated for 24 h with either 1 µM sodium arsenite or 1 nM estradiol in the presence or absence of 500 nM ICI 182,780. Progesterone receptor protein was determined using an enzyme immunoassay as described in Materials and Methods. Results are presented as femtomoles per mg protein (mean ± SD; n = 3).

 
Effect of arsenite on the steady state amount of pS2 mRNA
To determine whether arsenite regulates other estrogen-responsive genes, MCF-7 cells were treated with 1 µM arsenite for 24 h, and the amount of pS2 mRNA was measured by a RNase protection assay. In this study arsenite induced pS2 mRNA by 2.1-fold over control values (Fig. 4Go). Estradiol (1 nM) induced a 3.3-fold increase in pS2 mRNA. As in the case of the progesterone receptor, the effect of arsenite on pS2 mRNA was blocked by 500 nM ICI-182,780, suggesting that the effects of arsenite are mediated by ER{alpha}.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 4. Effect of arsenite on the steady state amount of pS2 mRNA. MCF-7 cells were grown and treated as described in Fig. 3Go. Total cellular RNA was extracted, and pS2 mRNA was determined by a RNase protection assay. Autoradiographs were quantified by phosphorimaging, and the values were expressed as the ratio of the pS2 signal to 36B4 signal. Results are presented as percentage of control values (mean ± SD; n = 4).

 
Interaction of arsenite with the hormone-binding domain of ER{alpha}
To demonstrate that arsenite activates ER{alpha}, a transient cotransfection assay was employed. A wild-type ER{alpha} expression vector and an estrogen response element-CAT reporter construct were cotransfected into COS-1 cells. The transfected cells were treated with concentrations of arsenite from 1 nM to 10 µM or with 1 nM estradiol. The amount of CAT activity was measured, expressed as the percent conversion, and normalized to the amount of ß-galactosidase activity (Fig. 5Go). As expected, estradiol stimulated CAT activity by approximately 4-fold. Arsenite, at concentrations as low as 1 nM, produced a 2-fold increase in CAT activity. Higher concentrations of arsenite increased CAT activity by 4-fold.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 5. The ability of arsenite to activate ER{alpha}. Wild-type ER{alpha} was transiently cotransfected with an estrogen response element-CAT construct into COS-1 cells. The transfected cells were treated for 24 h with 1 nM estradiol or with 1 nM to 10 µM sodium arsenite. CAT activity was measured as described in Materials and Methods. The results were normalized to ß-galactosidase and were expressed as a percentage of CAT activity in untreated cells (mean ± SD; n = 5).

 
To identify the region of ER{alpha} involved in activation by arsenite, chimeric receptors containing the hormone-binding domain of either ER{alpha} or the glucocorticoid receptor were employed. These chimeric receptors consist of the DNA-binding domain of the yeast transcription factor GAL-4 and the hormone-binding domain of either ER{alpha} (GAL-ER) or glucocorticoid receptor (GAL-GR). Stimulation of transcription by GAL-ER or GAL-GR from a GAL-4-responsive CAT reporter gene requires either estradiol or dexamethasone, respectively. When the chimeric receptor GAL-ER and the Gal-4-CAT reporter construct were transiently cotransfected into COS-1 cells and treated with 1 nM estradiol or 1 µM arsenite, there was an approximately 5-fold increase in CAT activity (Fig. 6Go). Addition of the antiestrogen ICI-82,780 (500 nM) had no effect on CAT activity, but blocked the effect of arsenite as well as the positive control estradiol. Arsenite has also been shown to block the binding of dexamethasone to glucocorticoid receptor (25). As arsenite interacts with the hormone-binding domain of the glucocorticoid receptor, GAL-GR was employed as a control, and the results are compared in Fig. 6Go. As expected, 100 nM dexamethasone induced an approximately 5-fold increase in CAT activity in cells transfected with GAL-GR. Arsenite had no effect and blocked the effect of dexamethasone. Taken together, these results suggest that arsenite activates ER{alpha} through an interaction with the hormone-binding domain of the receptor. Although arsenite has been shown to interact with the hormone-binding domain of GR, it does not activate transcription from a reporter gene construct.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 6. The ability of arsenite to activate GAL-ER. GAL-ER and GAL-GR chimeric genes and a GAL-4-CAT reporter construct were transiently cotransfected into COS-1 cells. The transfected cells were treated for 24 h with 1 nM estradiol, 100 nM dexamethasone, or 1 µM sodium arsenite in the presence or absence of 500 nM ICI-182,780. CAT activity was measured as described in Materials and Methods. The results were normalized to the ß-galactosidase activity and expressed as a percentage of the CAT activity in untreated cells (mean ± SD; n = 4).

 
Activation of ER{alpha} mutants by arsenite
Arsenite is capable of forming a coordination complex directly or indirectly with many different amino acids, including cysteines. The hormone-binding domain of ER{alpha} contains four cysteines at positions C381, C417, C447, and C530. To test the roles of these cysteines in the interaction with arsenite, each cysteine was mutated to alanine (29). The cysteine mutants C381A, C417A, C447A, and C530A as well as the quadruple mutant C381A C417A C447A C530A were then transiently cotransfected with an estrogen-responsive CAT construct into COS-1 cells, and the cells were treated with 1 nM estradiol or 1 µM arsenite. The amount of CAT activity was measured, expressed as the percent conversion, and normalized to the amount of ß-galactosidase activity (Fig. 7aGo). After treatment of the cysteine mutants with arsenite, there was an approximately 4-fold increase in CAT activity with mutants C417A and C530A. In contrast to the effects observed with these mutants, arsenite failed to activate the mutants C381A, C447A, and the quadruple mutant C381A C417A C447A C530A, suggesting that cysteines C381 and C447 may be involved in activation of ER{alpha} by arsenite. To demonstrate that the mutation of cysteine to alanine did not interfere with the activity of the receptor, the transiently transfected cells were treated with 1 nM estradiol. After hormone treatment, there was an approximately 4- to 5-fold increase in CAT activity with all mutants. These results corroborate previous studies employing these mutants, which demonstrate that mutation of cysteines in the hormone-binding domain to alanine does not alter the ability of estradiol to trans-activate the receptor (29).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 7. The ability of arsenite to activate wild-type and mutants of ER{alpha}. A, Wild-type ER{alpha}; the cysteine mutants C381A, C417A, C447A, and C530A; and the quadruple mutant C381A C417A C447A C530A were transiently cotransfected with an estrogen-responsive CAT construct into COS-1 cells. Transfected cells were treated for 24 h with either 1 nM estradiol or 1 µM sodium arsenite. CAT activity was measured as described in Materials and Methods. The results were normalized to the ß-galactosidase activity and were expressed as a percentage of the control value for wild-type ER{alpha} (mean ± SD; n = 3). B, Wild-type ER{alpha} and ER{alpha} mutants E523Q, D538N, H524A, K529Q.K531Q. N532D, K529Q.K531Q, and N532D were transiently cotransfected with an estrogen-responsive CAT construct into COS-1 cells, and the cells were treated as described above. CAT activity was measured. The results were normalized to the ß-galactosidase activity and were expressed as a percent of the control value for wild-type ER{alpha} (mean ± SD; n = 3). {square}, Control; {blacksquare}, estradiol; , arsenite.

 
To identify other possible interaction sites within the hormone-binding domain of ER{alpha}, histidine H524, lysines K529 and K531, and asparagine N532 were mutated (30, 31, 32). Glutamic acid E523 and aspartic acid D538 were mutated as negative controls. The ability of estradiol and arsenite to activate these mutants was also tested in transiently transfected COS-1 cells (Fig. 7BGo). Arsenite did not activate mutants H524A, K529Q K531Q N532D, K529Q K531Q, or N532D, but activated E523Q and D538N, resulting in an approximately 4-fold increase in CAT activity, suggesting that histidine H524, asparagine N532, and at least one of the lysines, K529 or K531, may also play a role in the interaction of arsenite with ER{alpha}. With the exception of H524, estradiol treatment of all mutants resulted in an approximately 4-fold increase in CAT activity, suggesting that mutation of these amino acids did not interfere with the activity of ER{alpha}. Estradiol treatment of H524A resulted in 2.8-fold induction of CAT activity, which is in agreement with previously published results (31).

Effect of arsenite on the binding of estradiol to ER{alpha}.
To determine whether arsenite blocks estradiol binding to ER{alpha}, the effects of the compound on hormone binding were measured using a single dose ligand binding assay. Cytosolic extracts from MCF-7 cells were treated on ice with various concentrations of arsenite (1 pM to 10 nM) for 1 h. The ability of ER{alpha} to bind hormone was then assayed by incubating the extract with 10 nM [3H]estradiol in the presence or absence of a 200-fold molar excess of diethylstilbestrol for 18 h at 4 C. As shown in Fig. 8Go, arsenite blocked the binding of estradiol to the receptor. Hormone binding decreased with increasing arsenite concentration. The inhibition constants (Ki) for arsenite was 5 ± 0.5 nM (n = 3), as determined by the method of Zhang and Danielsen (38). These results demonstrate that arsenite blocks the binding of estradiol to ER{alpha}. In contrast to arsenite, arsenate did not inhibit binding of estradiol to the receptor (data not shown), consistent with the charge differences between these compounds. Similar results were obtained when recombinant human ER{alpha} was used instead of MCF-7 cells (data not shown).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 8. Effect of arsenite concentration on estradiol binding to ER{alpha}. Cytosolic extracts from MCF-7 cells were treated for 1 h with various concentrations of sodium arsenite (1 pM; to 10 nM). The ability of ER{alpha} to bind hormone was assayed with 10 nM [3H]estradiol in the presence or absence of a 200-fold molar excess of diethylstilbestrol for 18 h at 4 C. The amount of specific binding of [3H]estradiol was determined as described in Materials and Methods and was expressed as a percentage of that in control cells (mean ± SD; n = 3).

 
Effect of arsenite on the growth of MCF-7 cells
The effect of arsenite on the anchorage-dependent growth of MCF-7 cells was also determined. Cells were treated with 1 nM estradiol or 1 µM sodium arsenite in the presence or absence of 500 nM ICI 182,780, and the number of cells was counted at different times. The results are presented in Fig. 9Go. Arsenite significantly stimulated the growth of MCF-7 cells compared with that of cells grown in estrogen-depleted medium. Growth stimulation induced by arsenite was similar to the growth stimulation induced by estradiol. The antiestrogen had no effect on cell growth and blocked arsenite- and estradiol-induced proliferation.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 9. Effect of arsenite on the growth of MCF-7 cells. MCF-7 cells were plated as described in Materials and Methods. After treatment with 1 µM sodium arsenite or 1 nM estradiol in the presence or absence of 500 nM of the antiestrogen ICI 182,780, cells were counted at different times (mean ± SD; n = 3). A, Effect of arsenite on the growth of MCF-7 cells. {blacksquare}, Control (C); •, estradiol (E2); {triangleup}, estradiol plus ICI; {diamondsuit}, arsenite (As); {diamond}, arsenite plus ICI.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we demonstrate that arsenite mimics the effects of estradiol in the ER{alpha}-positive human breast cancer cell line MCF-7. Similar to estradiol, arsenite decreased the expression of ER{alpha}, increased the expression of progesterone receptor and pS2, and activated ER{alpha} in transient transfection assays. More importantly, arsenite stimulated the growth of MCF-7 cells. The estrogen-like effects of arsenite were inhibited by an antiestrogen, suggesting that these responses were mediated by ER{alpha}. The most impressive effect was arsenite’s ability to activate ER{alpha} at concentrations as low as 1 nM. High affinity binding to ER{alpha} was demonstrated by the ability of arsenite to effectively block estradiol binding to the receptor (Ki = 5 nM), suggesting that the compound is more potent than most known environmental estrogens.

Arsenite has also been shown to activate signal transduction pathways and consequently regulate gene expression (39, 40, 41). Interestingly, the ability of arsenite to activate alternate signal pathways is concentration dependent. Low concentrations of arsenite induce extracellular signal-regulated kinase (ERK) phosphorylation and increase ERK activity, whereas high concentrations activate Jun kinase (40). We have recently shown that epidermal growth factor (EGF) also regulates ER activity in a concentration-dependent manner (42). High concentrations of EGF activate the ER in the absence of estradiol and enhance the activity of the receptor in the presence of hormone, whereas low concentrations of the growth factor do not activate the ER in the absence of estradiol and block hormone activation of the receptor. The ability of high and low concentrations of arsenite to activate different signal transduction pathways as well as activate the ER may explain the unusual dose-response curve observed in this study. High concentrations of arsenite, similar to high concentrations of EGF, may enhance the activity of the receptor, whereas low concentrations of arsenite may inhibit receptor activation, similar to low concentrations of EGF. The ability of arsenite to activate signal transduction pathways may also explain its ability to more effectively down-regulate ER expression than estradiol. Although estradiol and growth factors, such as EGF, transforming growth factor-ß, and insulin-like growth factor I, inhibit transcription of the ER gene (26, 35, 37, 42), the mechanisms by which they inhibit ER transcription appear to be different. The ability of arsenite to activate both mechanisms may explain its ability to more effectively decrease ER expression.

Binding assays and mutational analysis suggest that arsenite activates ER{alpha} through the formation of a high affinity interaction with the hormone-binding domain of the receptor. The compound competes with estradiol for binding to the receptor and activates a chimeric receptor containing the hormone-binding domain of ER{alpha}. Mutational analysis identified cysteines C381 and C447, histidine H524, asparagine N532, and at least one, and possibly two, lysines, K529 or K531, as potential sites consistent with the ability of arsenite to interact with amino acids containing either a thiol group or a positive charge. The hormone-binding domain of ER{alpha} contains 12 {alpha}-helixes (H1–H12) folded into a 3-layered antiparallel {alpha}-helical sandwich (43, 44, 45, 46, 47, 48). The central core layer contains 3 {alpha}-helixes (H5/6, H9, and H10) sandwiched between 2 additional layers of helixes composed of H1–4, H7, H8, and H11. The central core of the hormone-binding domain is flanked by helix H12. Upon binding of the ligand, a conformational change is induced, resulting in the formation of a salt bridge between H4 and H12 that repositions helix H12 over the central core and consequently entraps the hormone in a manner similar to a mouse trap (43). Ultimately, the repositioning of helix H12 results in the formation of a transcriptionally active receptor. The amino acids, identified as playing a role in the interaction of arsenite with ER{alpha}, are located on helixes H4, H8, and H11 and in the loop between H11 and H12. Cysteines 381 and 447 are located on helixes H4 and H8, respectively. Histidine 524 is located on helix H11 and is in close proximity to estradiol when the ligand is bound to the receptor. Asparagine N532 and lysines K529 and K531 are located in the loop between H11 and H12. It is possible that these amino acids participate directly in the formation of a metal-binding site or indirectly in the recruitment of arsenite to the binding site, i.e. arsenite may bind to the ER in a 2-step mechanism. In the first step of the latter model, it is postulated that arsenite would bind to H534, K529, K531, and N532 located on helix H11 and in the loop between helixes H11 and H12. The binding of arsenite would reposition helix H12 with respect to helix H11, resulting in the dissociation of proteins, such as heat shock proteins, from the receptor. In the second step, arsenite would bind to C381, C447, and possibly other amino acids and stabilize the active form of the receptor. Alternatively, in the first model, two molecules of arsenite would bind to the ER. One molecule would bind to C381 and C447, and the second molecule would bind to H524, K529, K531, and N532, resulting in a conformational change similar to the conformational change observed upon hormone binding. These models remain to be tested.

In previous studies (23, 24), we demonstrated that the heavy metal cadmium also mimics the effects of estradiol in estrogen-responsive breast cancer cells by a mechanism similar to that proposed for arsenite. Cadmium appears to activate ER{alpha} through the formation of a high affinity coordination complex with the hormone-binding domain of the receptor involving cysteines C381 and C447 and histidine H524. In contrast to the interaction of arsenite with positively charged amino acids, cadmium interacts with glutamic acid E523 on helix H11 and aspartic acid D583 at the loop-helix H12 interface, which is consistent with the ability of cadmium to form a coordination complex with negatively charged amino acids. The ability of cadmium as well as arsenite to bind and activate ER{alpha} suggests that these compounds may constitute a new class of environmental nonsteroidal estrogens.

The variation in national breast cancer incidence suggests that environmental factors play an important role in the etiology of the disease. In addition, the observation that the offspring of migrants from areas of low breast cancer incidence to areas of high breast cancer incidence acquire disease rates of the higher area provides further support for a role of the environment in the etiology of the disease. Although the environment appears to be an underlying cause of breast cancer, few environmental risk factors have been identified. The results of this study suggest that arsenite is a candidate environmental estrogen and therefore may pose a risk for breast cancer. Although this study suggests that exposure to arsenite may pose a risk, most studies to date have not implicated this compound in the etiology of the disease. Exposure to arsenite has been linked to carcinogenesis of the lungs, liver, kidney, bladder, skin, and, possibly, prostate. However, there is some experimental evidence to support a link between arsenic and breast cancer. In a spontaneous mouse model, low concentrations of arsenite in the drinking water stimulate the growth and increase the incidence of multiple mammary tumors (49), consistent with an estrogen-like promoter effect of arsenite. Although the principal exposure to arsenic in the general population occurs through drinking water, food, and cigarette smoke, populations residing near smelters may be exposed to higher levels of arsenic. In smelter workers, arsenic in the urine may be as high as 460 µg/liter, and in children living near smelters, arsenic in the urine is approximately 50 µg/liter (50). In pregnant women from areas of high and low contamination, maternal blood levels of arsenic range from 0.2–37.5 ng/ml, and placental concentrations range from 0.2–24 ng/ml (51). Breast tissue also contains significant amounts of arsenic, suggesting that arsenite could pose a risk for breast cancer. In addition to this, arsenic exposure has been linked to reproductive abnormalities. Although a number of studies have shown an increased number of spontaneous abortions and stillbirths and a decrease in birth weight in populations exposed to arsenic (52, 53), these populations are also exposed to other metals and chemicals associated with smelter activity, making it difficult to unequivocally link these abnormalities to arsenic exposure.

In summary, this study provides evidence that arsenite is a potential nonsteroidal environmental estrogen. In estrogen-responsive breast cancer cells, the compound mimicked the effects of estradiol, resulting in an increase in the steady state levels of progesterone receptor and pS2 and a decrease in the steady state level of ER{alpha}. Arsenite also stimulated cell proliferation, which was inhibited by an antiestrogen. The compound appears to activate ER{alpha} through the formation of a high affinity complex with the hormone-binding domain of the receptor, which blocks the binding of estradiol.


   Acknowledgments
 
We thank Dr. B. Katzenellenbogen and Prof. P. Chambon for providing estrogen receptor mutants, Dr. M. E. Lippman for helpful discussions, and Drs. L. Hilakivi-Clarke and M. Pentecost for critical reading of the manuscript.


   Footnotes
 
1 This work is supported by NIH Grant CA-7078 (to M.B.M.), the Cancer Research Foundation of America (to A.S.), the Susan G. Komen Foundation (to A.S.), and an anonymous donor. Back

Received February 15, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. American Cancer Society 1993 Cancer Facts and Figures—1993. American Cancer Society, Atlanta
  2. Jonnalagadda SB, Rao PV 1993 Toxicity, bioavailability and metal speciation. Comp Biochem Physiol C 106:585–595
  3. Klassen CD 1990 Heavy metals and heavy metal antagonists. In: Gilman AG, Rall TW, Nies AS, Taylor P (eds) Goodman and Gilman’s The Pharmacological Basis of Therapeutics. Pergamon Press, New York, pp 1592–1614
  4. Choiu H-Y, Hseuh Y-M, Liaw K-F, Horng S-F, Chaing M-H, Pu Y-S, Lin JSN, Huang C-H, Chen C-J 1995 Incidence of internal cancers and ingested inorganic arsenic: a seven-year follow-up study in Taiwan. Cancer Res 55:1296–1300[Abstract/Free Full Text]
  5. Enterline PE, Marsh GM 1982 Cancer among workers exposed to arsenic and other substances in a copper smelter. Am J Epidemiol 116:895–911[Abstract/Free Full Text]
  6. Enterline PE, Day R, Marsh GM 1995 Cancers related to exposure to arsenic at a copper smelter. Occup Environ Med 52:28–32[Abstract]
  7. Bates MN, Smith AH, Cantor KP 1995 Case-control study of bladder cancer and arsenic in drinking water. Am J Epidemiol 141:523–530[Abstract/Free Full Text]
  8. Chen CJ, Chen CW, Wu MM, Kuo TL 1992 Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. Br J Cancer 66:888–892[Medline]
  9. Mushak P, Crocetti AF 1995 Risk and revisionism in arsenic cancer risk assessment. Environ Health Perspect 103:684–689[Medline]
  10. Wu MM, Kuo T-L, Hwang Y-H, Chen C-J 1989 Dose response relation between arsenic concentration in well water and mortality from cancers and vascular diseases. Am J Epidemiol 130:1123–1132[Abstract/Free Full Text]
  11. Buchanan WE 1962 Toxicity of arsenic compounds. Elsevier, New York
  12. Das D, Chatterjee A, Mandel BK, Samanta G, Chanda B 1995 Arsenic in ground water in six districts of West Bengal, India: the biggest arsenic calamnity in the world. II. Arsenic concentration in drinking water, hair, nails, urine, skin-scale and liver tissue (biopsy) of the affected people. Analyst 120:917–924[Medline]
  13. Lewis DR, Southwick JW, Ouellet-Hellstrom R, Rench J, Calderon RL 1999 Drinking water arsenic in Utah: a cohort mortality study. Environ Health Perspect 107:359–365[Medline]
  14. Chen CJ, Wang CJ 1990 Ecological correlation between arsenic level in well water and age-adjusted mortality from malignant neoplasms. Cancer Res 50:5470–5474[Abstract/Free Full Text]
  15. Yeh S, Miyamoto H, Shima H, Chang C 1998 From estrogen to androgen receptor: A new pathway for hormone in prostate. Proc Natl Acad Sci USA 95:5527–5532[Abstract/Free Full Text]
  16. IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans: some metals and metallic compounds 1980 Lyon, France, vol. 23
  17. Pershagen G, Nordberg G, Bjorklund NE 1984 Carcinomas of the respiratory tract in hamsters given arsenic trioxide and benzo(a)pyrene by the pulmonary route. Environ Res 34:227[Medline]
  18. Lee T-C, Tanaka N, Lamb PW, Gilmer TM, Barrett JC 1988 Induction of gene amplification by arsenic. Science 241:79–81[Abstract/Free Full Text]
  19. Yamanaka K, Ohtsubo K, Hasegawa A, Ohji H, Kanisawa M, Okada S 1996 Exposure to dimethylarsinic acid, a main metabolite of inorganic arsenics, strongly promotes tumorrigenesis initiated by 4-nitozoline 1-oxide in the lungs of mice. Carcinogenesis 17:767–770[Abstract/Free Full Text]
  20. DiPaolo JA, Casto BC 1979 Quantitative studies of in vitro morphological transformation of Syrian hamster cells by inorganic metal salts. Cancer Res 39:1008–1013[Abstract/Free Full Text]
  21. Lee T-C, Oshimura M, Barrett JC 1985 Comparison of arsenic-induced cell transformation cytotoxicity, mutation and cytogenetic effects in Syrian hamster embryo cells in culture. Carcinogenesis 6:1421[Abstract/Free Full Text]
  22. Nordenson I, Beckman L 1991 Is the genotoxic effect of arsenic mediated by oxygen free radicals? Hum Hered 41:71–73[Medline]
  23. Garcia-Morales P, Saceda M, Kenney N, Kim N, Salomon DS, Gottardis MM, Solomon HB, Sholler PF, Jordan VC, Martin MB 1994 Effect of cadmium on estrogen receptor levels and estrogen-induced responses in human breast cancer cells. J Biol Chem 269:16896–16901[Abstract/Free Full Text]
  24. Stocia A, Katzenellenbogen BS, Martin MB 2000 Activation of estrogen receptor-{alpha} by the heavy metal cadmium. Mol Endocrinol 14:545–553
  25. Simons Jr SS, Chakraborti PK, Cavanaugh AH 1990 Arsenite and cadmium(II) as probes of glucocorticoid receptor structure and function. J Biol Chem 265:1938–1945[Abstract/Free Full Text]
  26. Saceda M, Lippman ME, Chambon P, Lindsey RK, Pongliktmongkol M, Puente M, Martin MB 1988 Regulation of the estrogen receptor in MCF-7 cells by estradiol. Mol Endocrinol 2:1157–1162[Abstract]
  27. Masiakowski P, Breathnach R, Bloch J, Gannon F, Krust A, Chambon P 1982 Cloning of cDNA sequences of hormone-regulated genes from MCF-7 human breast cancer cell line. Nucleic Acids Res 10:7895–7903[Abstract/Free Full Text]
  28. Chen C, Okayama H 1987 High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752[Abstract/Free Full Text]
  29. Reese JC, Katzenellenbogen BS 1991 Mutagensis of cysteines in the hormone binding domain of the human estrogen receptor. J Biol Chem 266:10880–10887[Abstract/Free Full Text]
  30. Wrenn CK, Katzenellenbogen BS 1993 Structure-function analysis of the hormone binding domain of the human estrogen receptor by region-specific mutagenesis and phenotypic screening in yeast. J Biol Chem 268:24089–24098[Abstract/Free Full Text]
  31. Pakdel F, Reese JC, Katzenellenbogen BS 1993 Identification of charged residues in an N-terminal portion of the hormone-binidng domain of the human estrogen receptor important in transcriptional activity of the receptor. Mol Endocrinol 7:1408–1417[Abstract]
  32. Pakdel F, Katzenellenbogen BS 1992 Human estrogen receptor mutants with altered estrogen and antiestrogen ligand discrimination. J Biol Chem 267:3429–3437[Abstract/Free Full Text]
  33. El-Ashry D, Chrysogelos SA, Lippman ME, Kern FG 1996 Estrogen induction of TGF-alpha is mediated by an estrogen response element composed of two imperfect palindromes. J Steroid Biochem Mol Biol 59:261–269[CrossRef][Medline]
  34. Webster NJG, Green S, Jin JR, Chambon P 1988 The hormone-binding domains of the estrogen and glucocorticoid receptors contain an inducible transcriptional activation function. Cell 54:199–207[CrossRef][Medline]
  35. Stoica A, Saceda M, Fakhro A, Solomon HB, Fenster BD, Martin MB 1997 The role of transforming growth factor-ß in regulation of estrogen receptor expression in the MCF-7 breast cancer cell line. Endocrinology 138:1498–1505[Abstract/Free Full Text]
  36. Laborda J 1991 36B4 cDNA used as an estradiol-independent messenger RNA control is the cDNA for human acidic ribosomal phosphoprotein P0. Nucleic Acids Res 19:3998[Free Full Text]
  37. Stoica A, Saceda M, Fakhro A, Joyner M, Martin MB 2000 Role of insulin-related growth factor-I in the regulation of estrogen receptor-alpha gene expression. J Cell Biochem 76:605–614[CrossRef][Medline]
  38. Zhang S, Danielsen M 1995 Selective effects of 8-Br-cAMP on agonists and antagonists of the glucocorticoid receptor. Endocrine 3:5–12
  39. Parrish AR, Zheng XH, Turney KD, Younis HS, Gandolfi AJ 1999 Enhanced transcription factor DNA binding and gene expression induced by arsenite or arsenate in renal slices. Toxicol Sci 50:98–105[Abstract/Free Full Text]
  40. Huang C, Ma WY, Li J, Goranson A, Dong Z 1999 Requirement of Erk, but not JNK, for arsenite-induced cell transformation. J Biol Chem 274:14595–14601[Abstract/Free Full Text]
  41. Chen W, Martindale JL, Holbrook NJ, Liu Y 1998 Tumor promoter arsenite activates extracellular signal-regulated kinase through a signalling pathway mediated by epidermal growth factor receptor and shc. Mol Cell Biol 18:5178–5188[Abstract/Free Full Text]
  42. Stoica A, Saceda M, Doraiswamy VL, Coleman C, Martin MB 2000 Regulation of estrogen receptor-alpha gene expression by epidermal growth factor. Endocrinology 165:371–378
  43. Wurta JM, Bourguet W, Renaud JP, Vivat V, Chambon P, Moras D, Gronemeyer H 1996 A canonical structure for the ligand-binding domain of nuclear receptors. Nat Struct Biol 3:87–94[CrossRef][Medline]
  44. Renaud JP, Rochel N, Ruff M, Vivat V, Chambon P, Gronemeyer H, Moras D 1996 Crystal structure of the RAR-{gamma} ligand-binding domain bound to all-trans retinoic acid. Nature 378:681–689
  45. Bourguet W, Ruff M, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-{alpha}. Nature 375:377–382[CrossRef][Medline]
  46. Brzozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson J, Carlquist M 1997 Molecular basis of agonism and antagonism in the estrogen receptor. Nature 389:753–758[CrossRef][Medline]
  47. Wagner RL, Apriletti JW, McGrath ME, West BL, Baxter JD, Fletterick RJ 1995 A structural role for hormone in the thyroid hormone receptor. Nature 378:690–697[CrossRef][Medline]
  48. Tanenbaum DM, Wang Y, Williams SP, Sigler PB 1998 Crystallographic comparison of the estrogen and progesterone receptor’s ligand binding domain. Proc Natl Acad Sci USA 95:5998–6003[Abstract/Free Full Text]
  49. Schrauzer GN, White DA, McGuinness JE, Schneider CJ 1978 Arsenic and cancer: effects of joint administration of arsenite and selenite on the genesis of mammary adenocarcinomas in inbred female C3H/St mice. Bioinorg Chem 9:245–253[CrossRef][Medline]
  50. Morse DL, Harrington JM, Housworth J, Landrigan MD 1979 Arsenic exposure in multiple environmental media in children near a smelter. Clin Toxicol 14:389–399[Medline]
  51. Kagey BT, Bumgarner JE, Creason JP 1977 Arsenic levels in maternal-fetal tissue sets. Trace Substances Environ Health 11:252–256
  52. Barlow SM, Sullivan FM 1982 Arsenic and its compounds. In: Reproductive Hazards of Industrial Chemicals. Academic Press, London, pp 62–82
  53. Hughes JP, Polissar L, Van Belle G 1988 Evaluation and synthesis of health effects studies of communities surrounding arsenic producing industries. Int J Epidemiol 17:407–413[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
D. J. Veselik, S. Divekar, S. Dakshanamurthy, G. B. Storchan, J. M.A. Turner, K. L. Graham, L. Huang, A. Stoica, and M. B. Martin
Activation of Estrogen Receptor-{alpha} by the Anion Nitrite
Cancer Res., May 15, 2008; 68(10): 3950 - 3958.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. B. Martin, R. Reiter, M. Johnson, M. S. Shah, M. C. Iann, B. Singh, J. K. Richards, A. Wang, and A. Stoica
Effects of Tobacco Smoke Condensate on Estrogen Receptor-{alpha} Gene Expression and Activity
Endocrinology, October 1, 2007; 148(10): 4676 - 4686.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
W. H. Watson and J. D. Yager
Arsenic: Extension of its Endocrine Disruption Potential to Interference with Estrogen Receptor-Mediated Signaling
Toxicol. Sci., July 1, 2007; 98(1): 1 - 4.
[Full Text] [PDF]

Home page
 Toxicol SciHome page
J. C. Davey, J. E. Bodwell, J. A. Gosse, and J. W. Hamilton
Arsenic as an Endocrine Disruptor: Effects of Arsenic on Estrogen Receptor-Mediated Gene Expression In Vivo and in Cell Culture
Toxicol. Sci., July 1, 2007; 98(1): 75 - 86.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
J. Kohrle, F. Jakob, B. Contempre, and J. E. Dumont
Selenium, the Thyroid, and the Endocrine System
Endocr. Rev., December 1, 2005; 26(7): 944 - 984.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
M. H. Herynk and S. A. W. Fuqua
Estrogen Receptor Mutations in Human Disease
Endocr. Rev., December 1, 2004; 25(6): 869 - 898.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
J. J. Pinzone, H. Stevenson, J. S. Strobl, and P. E. Berg
Molecular and Cellular Determinants of Estrogen Receptor {alpha} Expression
Mol. Cell. Biol., June 1, 2004; 24(11): 4605 - 4612.
[Full Text] [PDF]

Home page
 EndocrinologyHome page
M. B. Martin, R. Reiter, T. Pham, Y. R. Avellanet, J. Camara, M. Lahm, E. Pentecost, K. Pratap, B. A. Gilmore, S. Divekar, et al.
Estrogen-Like Activity of Metals in Mcf-7 Breast Cancer Cells
Endocrinology, June 1, 2003; 144(6): 2425 - 2436.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
W. H. Miller Jr., H. M. Schipper, J. S. Lee, J. Singer, and S. Waxman
Mechanisms of Action of Arsenic Trioxide
Cancer Res., July 15, 2002; 62(14): 3893 - 3903.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
E. K. Balcer-Kubiczek, G. H. Harrison, J.-F. Xu, and P. L. Gutierrez
Coordinate Late Expression of Trefoil Peptide Genes (pS2/TFF1 and ITF/TFF3) in Human Breast, Colon, and Gastric Tumor Cells Exposed to X-Rays
Mol. Cancer Ther., April 1, 2002; 1(6): 405 - 415.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow