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Endocrinology Vol. 143, No. 1 263-275
Copyright © 2002 by The Endocrine Society

RECEPTORS

Role of Cadmium in the Regulation of AR Gene Expression and Activity

Mary Beth Martin, H. James Voeller, Edward P. Gelmann, Jianming Lu, Elly-Gerald Stoica, Elijah J. Hebert, Ronald Reiter, Baljit Singh, Mark Danielsen, Elizabeth Pentecost and Adriana Stoica

Departments of Biochemistry and Molecular Biology (M.B.M., J.L., M.D., E.P., A.S.) and Oncology (M.B.M., H.J.V., E.P.G., E.-G.S., E.J.H., R.R., B.S., A.S.), Lombardi Cancer Center, School of Nursing and Health Studies (A.S.), Georgetown University, Washington, D.C. 20007

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Treatment of human prostate cancer cells, LNCaP, with cadmium stimulated cell growth. There was a 2.4-fold increase in the population of cells in the S + G2M phase by d 4 and a 2.7-fold increase in cell number by d 8. The metal decreased the concentration of AR protein and mRNA (80 and 60%, respectively) and increased the expression of prostate-specific antigen and the homeobox gene, NKX 3.1 (6-fold) that was blocked by an antiandrogen. In addition, cadmium activated the AR in mouse L cells containing an MMTV-luciferase reporter gene (4-fold increase) and in COS-1 cells transfected with wild-type AR and an MMTV-CAT reporter gene (7-fold increase). Cadmium also activated a chimeric receptor (GAL-AR) containing the hormone-binding domain of AR. The metal bound to AR with an equilibrium dissociation constant of 1.19 x 10-10 M. Cadmium blocked the binding of androgen to the receptor but did not alter its affinity (dissociation constant = 2.8 x 10-10 M), suggesting that the metal is an inhibitor of hormone binding. In castrated animals, a single, low, environmentally relevant dose of cadmium (20 µg/kg body weight) increased the wet weight of the prostate (1.97- to 3-fold) and the seminal vesicle complex (approximately 1.5-fold) and increased the expression of the androgen-regulated gene, probasin (27-fold). The in vivo effects were also blocked by an antiandrogen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTATE CANCER IS the most common cancer in men and the second leading cause of cancer-related deaths in American men (1). The risk factors for prostate cancer include age, endocrine status, genetic susceptibility, occupation, ethnicity, race, UV radiation, diet, and environmental factors (2). The importance of the latter in prostate cancer is supported by migration studies. When men from countries with a low prostate cancer incidence (Asia) migrate to the United States, a country with a high incidence of the disease, they acquire a risk that approaches the risk of American men (3, 4, 5). Although the factors responsible for the increased risk are not known, several studies suggest that exposure to the heavy metal cadmium may play a role in the etiology of the disease (6, 7, 8, 9, 10, 11, 12, 13).

The growth and development of the prostate gland is under the control of androgenic sex steroids (14, 15, 16). The effects of androgens are mediated by the AR, which regulates the expression of genes involved in the growth as well as in the secretory function of the gland. Androgens may also play a central role in the development of prostate cancer. A causal relationship between androgens and prostate cancer development is supported by the androgen-sensitivity and response to hormonal therapy of many prostate cancers (15, 16). Though the precise mechanism by which androgens affect prostate carcinogenesis is unknown, several hypotheses have been proposed. Androgens are strong promoters of carcinogenesis, acting via AR-mediated mechanisms, and may enhance the carcinogenicity of endogenous estrogen metabolites and estrogen- and prostatitis-generated reactive oxygen species and possible weak environmental carcinogens. All these processes seem to be modulated by a variety of environmental factors such as diet and life style habits, as well as by genetic determinants such as hereditary susceptibility and polymorphic genes that encode for steroid receptors and enzymes involved in the metabolism and activation of steroid hormones (15, 17, 18).

The AR is a member of a superfamily of genes that encode for steroid/thyroid hormone receptors. Members of the receptor family are ligand-dependent nuclear transcription factors, which have a similar modular structure (19). The N-terminal domain of the receptor is involved in the regulation of transcription and is the least conserved domain between members of the receptor family. The central region is a short, well-conserved cysteine-rich region, which corresponds to the DNA-binding domain. The C-terminal region of the receptor is the moderately conserved hormone-binding domain, which is composed of {alpha}-helices that form a ligand-binding pocket (20, 21, 22, 23, 24, 25, 26, 27).

Recent studies from this laboratory suggest that cadmium has potent estrogen-like activity. The metal binds with high affinity to the hormone-binding domain of ER-{alpha} and activates the receptor (28). The interaction of cadmium with ER-{alpha} involves several amino acids, some of which are also conserved in the hormone-binding domain of the AR (20, 25). In this study, we asked whether cadmium also mimics the effects of androgens by activating the AR and thereby poses a health risk.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue culture
The human prostate cancer cells, LNCaP (ATCC, Manassas, VA), 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. Cells were maintained in hormone-depleted medium for 2 d before treatment with cadmium chloride, zinc chloride, dihydrotestosterone (DHT) (Sigma, St. Louis, MO), methyltrienolone (R1881; NEN Life Science Products, Wilmington, DE), casodex (Zeneca Pharmaceuticals, Wilmington, DE), and hydroxyflutamide (Sigma).

For anchorage-dependent growth assays, LNCaP cells were plated at 105 cells/well into 6-well plates in IMEM supplemented with 10% charcoal-stripped serum. Cells were grown to 40% confluence, and the medium was changed to phenol red-free IMEM supplemented with 5% charcoal-stripped serum. After 2 d in this medium, cells were treated with either 10-9 M DHT or 10-6 M cadmium chloride. Cells were trypsinized at the specific time points and counted with a Coulter Counter (Coulter Electronics Inc., Hialeah, FL).

Cell cycle phase analysis
For cell cycle analysis, LNCaP cells were grown as described above and treated with either 10-9 M DHT or 10-6 M cadmium chloride. The cells were trypsinized to obtain a single cell suspension and verified microscopically after neutralizing with medium containing serum. The cell number was adjusted to 2 x 106 cells for each treatment. The cells were washed twice with PBS, centrifuged at 1000 rpm for 5 min, and suspended in 0.1 ml citrate/dimethylsulfoxide buffer. The DNA content was measured by Vindelov staining (29).

Western blot analysis
LNCaP cells were grown as described above and treated with either 10-9 M DHT, or 10-10–10-6 M cadmium chloride for 24 h in the presence or absence of the antiandrogen hydroxyflutamide (10-6 M). Cells were lysed in a buffer containing 20 nM Tris HCl (pH 9), 150 nM sodium chloride, 1% Nonidet P-40, 20 mM EDTA, 1 mM dithiothreitol, and the protease inhibitors pefabloc, aprotinin, and leupeptin. Lysates were loaded onto SDS-polyacrylamide gels. Gels were electrotransferred onto nitrocellulose membranes, washed in PBS five times at room temperature. Membranes were kept in blocking buffer overnight at 4 C and incubated for 1 h with 4 µg/ml of the polyclonal rabbit anti-AR antibody (Affinity BioReagents, Inc., Golden, CO) at room temperature. After three additional washes in PBS, membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:2000) in blocking buffer for 1 h at room temperature. Detection was performed by chemiluminescence using SuperSignal chemiluminescent substrate (Pierce Chemical Co., Rockford, IL).

AR hormone-binding assay
The ability of cadmium to compete with R1881 for binding to the AR was determined in extracts from LNCaP cells by a multiple-dose ligand-binding assay. LNCaP 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 (30). The protein concentration of the cell extract was determined by the Bradford method. Cell extracts were then pretreated with 10-6 M cadmium chloride for 1 h at 4 C; and concentrations of 17 {alpha}-methyl-[3H]R1881, ranging from 3 x 10-16 M to 3 x 10-9 M were added in the presence (nonspecific binding, BN) or absence (total binding, BT) of a 1000-fold molar excess of cold R1881. Two hours after incubating at 37 C, unbound steroid was removed by the addition of 5% dextran-coated charcoal. The amount of radioactivity was measured by scintillation counting. Total radioactivity was measured for each [3H]R1881 concentration, and specific binding (BS) was determined as the difference between BT and BN (BS = BT - BN). Data were plotted according to the method of Scatchard (31). The dissociation constant (Kd) and binding capacity were determined. The AR concentration was calculated as binding capacity divided by the total protein concentration and was expressed as femtomoles per milligram protein.

The ability of cadmium to bind to the AR was determined in cell extracts from LNCaP cells or COS-1 cells transiently transfected with the AR gene, by a multiple-dose ligand-binding assay. Cell extracts were incubated with various concentrations of 109Cd (10-12–10-6 M), in the presence or absence of a 1000-fold molar excess of R1881, for 2 h at 37 C. Free radioactivity was removed by the addition of 5% dextran-coated charcoal, and the amount of 109Cd bound to the AR was measured by scintillation counting. The data were analyzed by the method of Scatchard (31).

Measurement of AR, prostate-specific antigen (PSA), and NKX3.1 mRNA
Total cellular RNA was extracted using the RNazol method. The amounts of AR, PSA, and NKX3.1 mRNA were determined by a ribonuclease (RNase) protection assay. 32P-Labeled antisense RNA (cRNA) was synthesized in vitro from AR-2 (32), PSA (32), and acidic ribosomal phosphoprotein PO (p36B4) (33) using T7 polymerase, and from the human homeobox gene NKX3.1 (34) using T3 polymerase. The AR probe used for hybridization was a 713-bp EcoRI/HindII fragment covering nucleotides 1850–2563 of the human AR cDNA (32). Sixty micrograms of total RNA were hybridized, for 16 h at 50 C, to the 32P-labeled cRNAs. The samples were digested with RNase A for 30 min at 25 C. The protected cRNA probes were resolved on 6% polyacrylamide gels. The bands were visualized by autoradiography and quantified by phosphor imaging (Molecular Dynamics, Inc., Sunnyvale, CA). The amounts of AR, PSA, and NKX3.1 mRNAs were normalized to the amount of acidic ribosomal phosphoprotein PO mRNA.

Transient transfections and chloramphenicol acetyl transferase (CAT) assays
A low-temperature and low-pH calcium phosphate method was employed to transfect COS-1 cells (35). COS-1 cells were plated, at a density of 3 x 106 cells/150-mm dish, in phenol red-free IMEM containing 10% charcoal-treated calf serum, for 24 h. The cells were transfected with 120 µg DNA containing 15 µg of an AR expression vector or the chimeric receptor, GAL-AR (amino acids 624–918) (36), 75 µg of the reporter construct MMTV-CAT or a GAL4-CAT reporter construct (17m2GCAT), respectively, 6 µg of ß-galactosidase, and salmon sperm DNA. The LipofectAMINE (Life Technologies, Inc./BRL, Rockville, MD) method was used to cotransfect COS-1 cells with wild-type AR or the chimeric receptor GAL-AR and MMTV-CAT or 17M2GCAT, respectively (37), in the presence or absence of the coactivator ARA70 (AR-associated protein 70). Cells (106) were plated into 100-mm dishes and transfected with 2 µg of the AR expression vector, wild-type or GAL-AR; 10 µg of the reporter construct MMTV-CAT or 17m2GCAT, respectively; and 2, 4, 5, 6, or 10 µg of the coactivator AR-associated protein 70 (ARA70). 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 10-9 M DHT, 10-6 M cadmium chloride, or 10-5 M zinc chloride. The cells were harvested, 24 h later, and CAT activity was measured as described previously (38). 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 untreated controls.

Stable transfections and luciferase assays
Mouse L cells, L929, were cultured in DMEM supplemented with 3% calf serum. The day before transfection, 106 cells were plated on 100-mm tissue culture dishes. The calcium phosphate method (35) was used to transfect 20 µg MMTV-luciferase and 0.5 µg pSV2-neo into the L cells. After transfection, the cells were washed using PBS, and the medium was replaced with DMEM containing 3% calf serum for 24 h. Stably transfected cells were selected in DMEM supplemented with 3% calf serum and 500 µg/ml G418 for about 1 month. Colonies were isolated and treated with 10-9 M DHT, 10-6 M cadmium chloride, and 10-5 M zinc chloride. The amount of luciferase activity was determined using the luciferase assay system kit (Promega Corp., Madison, WI) as described by the manufacturer.

Animal studies
Male Wistar [Hanover and Hsd: (WI) BR] rats and C57BL/6-TgN mice were obtained from Harlan Inc. (Indianapolis, IN) and The Jackson Laboratory (Bar Harbor, MN), respectively. Animals were housed, three to a cage, in a conventional, well ventilated animal room kept at 22 ± 2 C and 40–70% relative humidity on a 12-h light, 12-h dark cycle. The animals had free access to water and Purina diet (Quality Lab Products, Elkridge, MD). At approximately 8 wk of age, the rats or mice were castrated either surgically or chemically using cyproterone acetate (CPA, Sigma). CPA was dissolved in 20% benzoylbenzoate in peanut oil at a concentration of 85 mg/ml. Animals were given ip injections of CPA (50 mg/kg body weight) daily for 18 d.

To determine the effect of cadmium chloride on the wet weight of the prostate and the seminal vesicle complex (seminal vesicles with coagulating glands), castrated rats or mice were randomly placed into eight groups. Each group contained five to eight animals. After a 7-d recovery from castration, each group was treated as indicated below. One group of animals did not receive treatment (control). A testosterone propionate pellet (100 mg/pellet, 112-d release (Innovative Research of America, Sarasota, FL) was sc implanted into the intrascapular region of two groups of animals under methoxyflurane anesthesia. Two groups of animals received a single dose of cadmium chloride (10 µg/kg; ip injection) prepared in PBS, whereas two additional groups received two doses of cadmium chloride (2 x 10 µg/kg) over 2 d (ip injection). Groups that were treated with an antiandrogen received ip injections of CPA (50 mg/kg body weight) daily for 10 d. Ten days after the start of treatment, the rats or mice, as well as four intact animals, were killed using CO2. The prostate, seminal vesicles with the coagulating glands, testes, livers, and kidneys were removed, freed from fat tissue, and weighed. All organs were fixed and prepared for histological examination. All animal studies were conducted in accord with the Guidelines for Care and Use of Experimental Animals.

Real-time PCR assay
Total RNA was isolated from the prostate glands with RNA STAT-60 (Tel-Test, Friendswood, TX). Twenty micrograms of RNA were suspended in 10 µl of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 20 mM MgCl2 containing 2 U deoxyribonuclease and were incubated at 37 C for 45 min. Deoxyribonuclease was heat inactivated before the addition of 50 U MuLV reverse transcriptase, 20 pmol random hexamers, 20 U RNase inhibitor, 10 nM deoxynucleotide triphosphate, and 2 µl RT buffer. The samples were incubated for 10 min at 25 C, 60 min at 37 C, and 5 min at 95 C. The RT product was diluted to a final vol of 200 µl in sterile water. For real time PCR, the iCycler iQ Detection System (Bio-Rad Laboratories, Inc., Hercules, CA) was used. Amplification was performed in a 25-µl final vol containing 1x reaction buffer (SYBR Green PCR core reagent kit; PE Applied Biosystems, Foster City, CA), 3 mM MgCl2, 0.25 µl Platinum Taq polymerase (Life Technologies, Inc., Carlsbad, CA), 0.2 mM deoxynucleotide triphosphate, 2 µl cDNA, and 0.5 µM primers. The oligonucleotide primers used to detect rat probasin were 5'-GGACCTACTTCCGTCGCATT-3' and 5'-ACGTCTTGGGATCTCCTTCC-3'. The primers used to detect rat glyceraldehyde 3-phosphate dehydrogenase were 5'-TCCTGCACCACCAACTGCTTAG-3' and 5'-CAGATCCACAACGGATACATTGG-3'. The PCR reaction conditions were: 10 min at 95 C, followed by 50 cycles of 20 sec at 95 C, 30 sec at 58 C, and 30 sec at 72 C. Fluorescent data were collected during the 72-C step.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of cadmium on the growth of LNCaP cells
Previous studies from our laboratory demonstrated that the heavy-metal cadmium binds to and activates ER-{alpha} (28, 38). The goal of the present study was to determine whether the metal binds to and activates the AR. To examine the androgen-like effects of cadmium, the human prostate cancer cell line, LNCaP, was chosen because it is the only human prostate cell line that demonstrates hormone-dependent growth (39). However, the AR in this cell line contains a point mutation at amino acid 877 (thr to ala) (40). We have previously shown that cadmium sulfate does not stimulate the growth of LNCaP cells (32). Because cadmium sulfate is a weak salt (Kd = 5 x 10-3 M), cadmium chloride was employed. To prevent metallothionein induction, 10-6 M cadmium chloride was used (41). In the first study, the effect of cadmium on anchorage-dependent growth was determined. Cells were treated with either 10-9 M DHT or 10-6 M cadmium chloride in the presence or absence of the antiandrogen casodex (10-6 M). The number of cells was counted on subsequent days, and the results are presented in Fig. 1Go. As expected, DHT stimulated the growth of LNCaP cells. There was a 3.2-fold increase in cell number on d 8 (P < 0.0001), when compared with cells grown in androgen-depleted medium. Cadmium also stimulated the growth of LNCaP cells; there was a 2.7- fold increase in cell number (P < 0.0001). The effects of DHT and cadmium on growth were additive (5.3-fold induction, P < 0.0001). The antiandrogen blocked the cadmium-induced cell proliferation, suggesting that the effects of the metal were mediated by the AR.



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  		Figure 1. Effect of cadmium on the growth of LNCaP cells. LNCaP cells were plated and grown to 40% confluence. The medium was changed to phenol red-free medium supplemented with 5% charcoal-stripped serum for 2 d. Cells were treated with either 10-9 M DHT or 10-6 M cadmium chloride in the presence or absence of 10-6 M casodex (cas). After treatment, cells were counted at different time points. Results represent the mean value of three experiments ± SD.

 
To further characterize the mitogenic effects of cadmium on growth, cell cycle analysis was performed, and the results are presented in Table 1Go. After 4 d of treatment with either 10-9 M DHT or 10-6 M cadmium chloride, the population of cells in the S + G2M phases of the cell cycle increased by 1.8-fold in DHT-treated cells (P = 0.0031) and by 2.4-fold in cadmium treated cells (P = 0.001), suggesting that both DHT and cadmium induced significant proliferation.


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  		Table 1. Effect of DHT and cadmium on cell cycle analysis (% cells in that phase of the cell cycle)

 
Effect of cadmium on AR protein concentration
To determine the effect of cadmium on the expression of AR protein, a Western blot was employed. The LNCaP cells were treated for 24 h with 10-9 M DHT, 10-10–10-6 M cadmium chloride, or 10-6 M cadmium chloride plus an antiandrogen (10-6 M CPA or hydroxyflutamide). Cells were lysed, and Western blot analysis was performed using a polyclonal rabbit anti-AR antibody (Fig. 2AGo). The amount of AR was quantified, and data are presented in Fig. 2BGo as percent of untreated control. The results demonstrate a decrease in AR protein when cells were treated with increasing concentrations of cadmium. The amount of AR protein decreased 37% after treatment with 10-10 M cadmium, 52% with 10-8 M cadmium, and 79% with 10-6 M cadmium. Addition of CPA or hydroxyflutamide blocked the effect of cadmium. Similar to other studies in LNCaP cells, DHT did not significantly decrease AR protein (11%, P = 0.051, as measured by t test). The ability of DHT to decrease AR mRNA expression, but to have no effect on AR protein expression in LNCaP cells, has been previously observed (42). To determine whether changes in the amount of AR protein correlated with changes in the number of binding sites, the number of sites was determined using a single-dose ligand-binding assay (Fig. 2CGo). Whereas treatment with DHT did not significantly decrease the number of AR-binding sites, treatment with cadmium decreased the number of binding sites by 20–50% (from 861 fmol/mg protein in control cells to 689, 517, and 431 fmol/mg protein in cells treated with 10-10 M, 10-8 M, and 10-6 M cadmium chloride, respectively). These results demonstrate a similar decrease in AR protein and binding sites after treatment with cadmium. The cadmium-induced decrease in the number of binding sites was also blocked by 10-6 M hydroxyflutamide. There was no statistically significant difference between control and cadmium plus hydroxyflutamide treatment (P = 0.0307), suggesting that the effect of cadmium was mediated by the AR.



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  		Figure 2. Effect of cadmium on AR protein. LNCaP cells were grown, as described in Fig. 1Go, and treated for 24 h with 10-9 M DHT, 10-12 –10-6 M cadmium chloride, or 10-6 M cadmium chloride plus 10-6 M hydroxyflutamide (OHF) or CPA. A, Western blot analysis was performed using a polyclonal rabbit anti-AR antibody. A representative blot from three experiments is shown. B, Western blots were quantified, and the data were presented as percent of nontreated controls. C, The number of AR-binding sites were determined by a single-dose (10-8 M R1881) ligand-binding assay. Results are expressed as percent of control cells and represent the mean value of three experiments ± SD. OHF, Hydroxyflutamide.

 
Effect of cadmium on the steady-state concentration of AR mRNA
An RNase protection assay was performed to determine whether cadmium altered the amount of AR mRNA in the cell. Cells were treated for 24 h with either DHT or cadmium chloride. Total RNA was isolated and hybridized to a 32P-labeled antisense riboprobe for the human AR. After digestion with RNase A, the protected fragments were resolved on a 6% polyacrylamide gel. The bands were quantified by scanning densitometry and normalized to the amount of acidic ribosomal protein PO, which is constitutively expressed in the presence of cadmium (38). In contrast to the effects of the androgen on the amount of AR protein, treatment with 10-9 M DHT produced a decrease of approximately 60% in the amount of AR mRNA that was blocked by 10-6 M casodex (Fig. 3AGo). Treatment for 24 h with 10-10–10-6 M cadmium produced a 50–60% decrease in the amount of AR mRNA (Fig. 3AGo), which correlates with the effect of the metal on AR protein concentration. Similar to the effects on AR protein, the effects of cadmium on AR mRNA were also blocked by the antiandrogens casodex and hydroxyflutamide. A time course of the response to 10-6 M cadmium is presented in Fig. 3BGo. Cadmium produced a rapid decrease in AR mRNA; by 1 h, a 40% decrease was observed. The maximum decrease of approximately 60% was observed at 18 h, and the amount of AR mRNA remained suppressed for 48 h. Taken together, these results provide additional support that the effects of cadmium are mediated by the AR.



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  		Figure 3. Effect of cadmium on AR mRNA. LNCaP cells were grown, as described in Fig. 1Go, and treated with 10-9 M DHT or 10-12–10-6 M cadmium chloride in the presence or absence of 10-6 M casodex or hydroxyflutamide. Total mRNA was prepared, and AR mRNA was determined by an RNase protection assay as described in Materials and Methods. The effects of cadmium on AR mRNA were normalized to the amount of acidic ribosomal phosphoprotein PO mRNA and expressed as percent of control values. Data represent the mean value of four experiments ±SD. A, Effect of cadmium concentration on AR mRNA; B, time course of the effect of cadmium on AR mRNA.

 
Effect of cadmium on the activity of the AR
To further characterize the effects of cadmium on the activity of the AR, the ability of the metal to induce two androgen-regulated genes, PSA (32) and the human homeobox gene NKX3.1 (34), was measured. The LNCaP cells were treated for 24 h with cadmium chloride concentrations from 10-12–10-6 M in the presence or absence of 10-6 M casodex. The synthetic androgen R1881 (10-9 M) was used as a positive control. After treatment, an RNase protection assay was performed, and the results are presented in Fig. 4Go, A and B. As expected, R1881 increased PSA and NKX3.1 mRNA by 7- and 5.7-fold, respectively. Cadmium increased PSA mRNA and NKX 3.1 mRNA by 2- to 6-fold in a concentration-dependent manner. Casodex blocked the induction by either R1881 or cadmium, providing additional evidence that the effects of the metal were mediated by the AR.



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  		Figure 4. Effects of cadmium on AR activity. A, Effect of cadmium on PSA mRNA. B, Effect of cadmium on NKX3.1 mRNA. LNCaP cells were grown as described in Fig. 1Go. Cells were treated for 24 h with 10-9 M R1881 or 10-12–10-6 M cadmium chloride in the presence or absence of 10-6 M casodex. The amounts of PSA and NKX3.1 mRNA were determined by an RNase protection assay. The data were normalized to the amounts of acidic ribosomal phosphoprotein PO mRNA, and the results were expressed as percent of control values. C, COS-1 cells were transiently cotransfected with a wild-type human AR expression vector and the MMTV-CAT reporter construct. After transfection, the cells were treated with 10-9 M DHT, 10-12–10-6 M cadmium chloride, or 10-5 M zinc chloride for 24 h. Transfected cells were harvested and assayed for CAT activity, and the results were normalized to ß-galactosidase. D, Mouse L cells, which express the endogenous AR gene and contain a stably transfected MMTV-luciferase reporter construct, were treated for 24 h with 10-9 M DHT, 10-6 M cadmium chloride, and 10-5 M zinc chloride. Luciferase activity was measured and normalized to ß-galactosidase activity. Data represent the mean value of three experiments ± SD.

 
Because the AR in LNCaP cells contains a mutation at amino acid 877 in the hormone-binding domain that alters steroid specificity and antiandrogen sensitivity, the effects of cadmium on wild-type AR activity were tested in two different transfection assays. In the first assay system, COS-1 cells were transiently cotransfected with a wild-type human AR expression vector and the mouse mammary tumor virus-CAT reporter gene. In the second assay system, mouse L cells, which express wild-type AR, were stably transfected with the mouse mammary tumor virus-luciferase reporter construct. Transfected cells were treated with 10-9 M DHT, 10-12–10-6 M cadmium chloride, or 10-6–10-5 M zinc chloride for 24 h, and CAT or luciferase activity was measured (Fig. 4Go, C and D). In COS-1 cells transfected with the wild-type human AR, cadmium increased the amount of CAT activity by 3- to 10-fold (Fig. 4CGo). In cells that express the endogenous mouse AR, cadmium chloride treatment resulted in a 4-fold increase in luciferase activity (Fig. 4DGo). Treatment with DHT increased CAT activity by 15-fold, and luciferase activity by 10-fold. However, zinc [10-6 M (data not shown) or 10-5 M] was not capable of increasing either CAT or luciferase activity. The ability of cadmium to transactivate AR in these assays demonstrates that the metal also activates wild-type human and mouse AR, and this effect is specific for cadmium and not for zinc.

One of the AR-associated proteins that contributes to the activation of AR is ARA70 (36). To determine whether ARA70 can enhance the transcriptional activity of AR in the presence of cadmium, COS-1 cells were transiently cotransfected with wild-type human AR, the mouse mammary tumor virus-CAT reporter, and increasing amounts of ARA70 from 2–6 µg (Fig. 5Go). ARA70 did not change the baseline CAT activity. However, the coactivator enhanced the DHT-induced transcriptional activity of AR in a dose-dependent manner. After treatment with DHT, CAT activity increased by 19-, 34-, and 75-fold in the presence of 2, 4, and 6 µg ARA70, respectively. A similar enhancement of AR activity was observed after cadmium treatment. In the absence of added ARA70, a 6-fold induction of CAT activity was measured. With increasing amounts of ARA70, cadmium induced CAT activity by 19-, 29-, and 53-fold, suggesting that ARA70 enhances AR activity when the receptor is activated by both androgens and cadmium.



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  		Figure 5. Effect of cadmium on ARA70 stimulation of AR transcriptional activity. COS-1 cells were transiently cotransfected with 2 µg of an AR expression vector and 10 µg of the MMTV-CAT reporter construct in the presence or absence of 2, 4, or 6 µg of the coactivator ARA70. Transfected cells were treated for 24 h with either 10-9 M DHT or 10-6 M cadmium chloride, and CAT activity was determined as described in Materials and Methods. The amount of CAT activity was normalized to the amount of ß-galactosidase activity. Results are expressed relative to CAT activity of cells in the absence of ARA70. The results are the mean value of three experiments ± SD.

 
Interaction of cadmium with the hormone-binding domain of the AR
To identify the region of AR involved in activation by cadmium, a chimeric receptor (GAL-AR) containing the DNA-binding domain of the yeast transcription factor GAL4 and the hormone-binding domain of AR from amino acids 624–918 (GAL-AR) (36) was employed. Stimulation of transcription by GAL-AR from a GAL4-responsive reporter gene requires the presence of a coactivator and DHT (43). In the absence of ARA70, GAL-AR did not activate GAL4-CAT in response to treatment with either 10-9 M DHT or 10-6 M cadmium chloride. However, when the coactivator ARA70 was coexpressed with GAL-AR, an increase in CAT activity was observed in the presence of DHT or cadmium (Fig. 6Go). After treatment with DHT, CAT activity increased by 4- (P < 0.0001) and 5.5-fold (P = 0.0004) in the presence of 5 and 10 µg ARA70, respectively. Cadmium also activated the GAL-AR receptor. There was a 2.5- (P = 0.146) and 3.5-fold (P = 0.0065) increase in CAT activity in the presence of 5 and 10 µg ARA70, respectively. However, zinc did not activate GAL-AR, nor did it block the effects of cadmium. Neither DHT nor cadmium activated a mutant receptor containing only the DNA-binding domain of GAL4 (data not shown). The ability of cadmium to activate the GAL-AR chimeric receptor suggests that cadmium activates AR through the hormone-binding domain.



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  		Figure 6. Interaction of cadmium with the hormone-binding domain of the AR. COS-1 cells were transiently transfected with GAL-AR and a GAL4-CAT reporter plasmid in the presence or absence of the coactivator ARA70. The transfected cells were treated with 10-9 M DHT, 10-6 M cadmium chloride, or 10-5 M zinc chloride in the presence or absence of the antiandrogen casodex (10-6 M). CAT activity was measured as described in Materials and Methods. The results were normalized to the ß-galactosidase activity and expressed as percent of CAT activity in untreated cells. The results represent the mean value of three experiments ± SD.

 
Binding of cadmium to the AR
To determine whether cadmium competes with androgen for binding to the AR, the effects of the heavy metal on hormone binding were measured using a multiple-dose ligand-binding assay. Cell extracts from LNCaP cells were preincubated with 10-6 M cadmium chloride for 1 h at 4 C. Radiolabeled R1881 was added in the presence or absence of a 1000-fold molar excess of cold R1881 for 2 h at 37 C. The affinity and binding capacity of the receptor were determined according to the method of Scatchard (Fig. 7AGo). A 45% decrease in the number of androgen-binding sites from 835 to 459 fmol/mg protein was observed after addition of 10-6 M cadmium chloride. The Kd of the [3H]R1881-AR complex was (2.43 ± 0.35) x 10-10 M (n = 3, r = 0.87) and did not show any significant change in the presence of cadmium, (2.75 ± 0.43) x 10-10 M (n = 3, r = 0.73). These results indicate that the metal does not alter the binding affinity of R1881 to the receptor but blocks hormone binding to the AR.



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  		Figure 7. Binding of cadmium to the AR. LNCaP cells were grown in IMEM supplemented with 5% FCS. At 70% confluence, medium was changed to phenol red-free IMEM and 5% charcoal-treated serum. Cells were grown in this medium for 2 d. The ligand-binding assay was performed in cell extracts as described in Materials and Methods. The binding affinity and binding capacity were determined by the method of Scatchard as described in Materials and Methods. Experiments were repeated three times ± SD. A, Competition between cadmium and R1881 for binding to AR. B, Binding of cadmium to AR.

 
To determine the binding affinity of cadmium, a multiple-dose ligand-binding assay using 109Cd was employed. Cell extracts from LNCaP cells were incubated with 109Cd (10-12–10-6 M) in the presence or absence of a 1000-fold molar excess of R1881 for 2 h at 37 C. The Scatchard plot is represented in Fig. 7BGo. Cadmium bound AR with high affinity, Kd = (1.19 ± 0.55) x 10-10 M (n = 3, r = 0.90) and a binding capacity of 655 ± 81 fmol/mg protein, suggesting the existence of a high-affinity binding site for cadmium in the LNCaP cell extracts. To show that the high-affinity site was the AR, COS-1 cells were transiently transfected with the AR gene, and the ability of 109Cd to bind to extracts from control and transfected cells was measured. High-affinity binding (Kd = 2 x 10-10 M, data not shown) was observed in COS-1 cells, whereas no specific binding was observed in control cells, suggesting that cadmium binds with high affinity to the AR.

In vivo effects of cadmium
Both the functional and the structural maintenance of prostate are dependent on androgens, and the prostate rapidly regresses after withdrawal of androgen support, such as with castration (14, 44, 45). To determine whether cadmium mimics androgenic responses in animals, the effects of the metal on the wet weight of the prostate and seminal vesicle complex (seminal vesicles and coagulating glands) were tested in castrated animals. The total dose of cadmium chloride was either 10 or 20 µg/kg body weight (~54 or 108 nmol/kg, respectively). These doses are 1/250 and 1/500 of the LD50 of the metal and are equivalent to the daily exposure (23 µg/kg body weight per day) from food and drinking water (46, 47). T propionate, a mitogen in rat prostate and an effective promoter of prostate cancer in rodents (48), was employed as a positive control. In the first study, male Wistar rats (Hanover) were surgically castrated at 8 wk of age. After a 7-d recovery, the animals received either one or two ip injections of cadmium chloride (10 µg/kg body weight) or T propionate (1 mg/kg·d). In addition to cadmium and T, some experimental animals received CPA (50 mg/kg·d for 10 d, ip injection). The effects of cadmium on the average weight of prostate and seminal vesicle complexes are shown in Table 2Go. Although a small decrease (10–15%, P < 0.05) in the average body weight was observed when the animals were castrated, there was no statistical difference in body weight between treatments. Atrophy of the prostate and seminal vesicle complexes was observed in castrated animals, compared with intact rats. As expected, T increased the wet weight of the prostate by 10.8-fold (P < 0.0001), and the weight of the seminal vesicles by 9.7-fold (P < 0.0001), when compared with nontreated castrated animals (Table 2Go). These results are in agreement with an earlier report (45). As expected, treatment with the antiandrogen blocked the effects of the androgen. A single dose of cadmium also induced a 1.61- (P = 0.0014) and 1.43-fold (P < 0.001) increase in the wet weight of prostate and seminal vesicle complexes, respectively, whereas two doses of cadmium produced a 1.97- (P = 0.0002) and 1.65-fold (P < 0.0001) increase in the wet weight of prostate and seminal vesicle complexes, respectively. When rats were treated with cadmium and the antiandrogen, the effects of the heavy metal on the increase in wet weight of the prostate and seminal vesicles was blocked, suggesting that the effects of cadmium are mediated by AR. Histological examination (Fig. 8Go) of the prostate from castrated animals showed atrophic glands lined by small columnar cells and no papillae (i.e. infoldings of the epithelium), whereas the glands from T-treated animals were back-to-back with numerous papillae and were lined by stratified columnar epithelium with abundant cytoplasm. The glands from cadmium-treated animals also showed epithelial proliferation and numerous papillae. When animals were treated with cadmium and the antiandrogen, a simple architecture of the gland was observed.


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  		Table 2. Effects of cadmium on the wet weight of androgen target organs

 


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  		Figure 8. Histological effects of cadmium in the prostate gland of orchiectomized animals. Male Wistar rats (Hanover), castrated at 8 wk of age, were treated with T propionate (1 mg/kg·d) or two doses of cadmium chloride (2 x 10 µg/kg) in the presence or absence of the antiandrogen CPA (50 mg/kg·d) for 10 d. Control, Prostate shows small atrophic glands lined by small columnar cells. No papillae are noted. Testosterone, The glands are back-to-back with numerous papillae and are lined by stratified columnar epithelium with abundant cytoplasm. Cadmium, The glands show epithelial proliferation with numerous papillae. Cadmium plus CPA, The architecture of the glands is simplified.

 
In addition to increasing the wet weight of the prostate and seminal vesicle complex, cadmium induced the expression of the androgen-regulated gene, probasin (Fig. 9Go). There was a 21- to 27-fold increase in probasin mRNA in the prostates of animals treated with either one or two doses of cadmium, respectively. The increase in probasin was also blocked by the antiandrogen, providing additional evidence that the effects of cadmium are mediated by the AR.



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  		Figure 9. Effect of cadmium on probasin expression. Male Wistar animals were treated as in Fig. 8Go. The amount of probasin mRNA was determined by real-time PCR and normalized to the amount of glyceraldehyde 3-phosphate dehydrogenase. The results represent the mean value of three animals ± SD.

 
Because chemical castration, followed by treatment with androgen, enhances proliferation in the prostate (49), a second study was conducted. Male Wistar rats (Hanover) were chemically castrated by treating with the antiandrogen CPA (50 mg/kg body weight·d, ip injection) for 18 d, beginning at 8 wk of age. After a 7-d recovery, the animals were placed in the same eight groups and treated as in the previous experiment. Similar to surgical castration, a single dose of cadmium produced a 1.8-fold increase in prostate wet weight; however, there was no significant effect on the wet weight of the seminal vesicle complex. In contrast to a single dose of cadmium, two doses of the heavy metal produced a 2.5-fold (P < 0.0001) increase in prostate wet weight and a statistically significant increase in the wet weight of the seminal vesicle complexes (1.8-fold, P < 0.0001). Treatment with the antiandrogen also blocked the effects of either androgen or cadmium, suggesting that the effects of the metal are mediated by AR. The weights of the testes were similar in all groups, independent of treatment. Histological examination of the prostate, seminal vesicles, coagulating glands, testes, kidney, and liver demonstrated no metal toxicity (data not shown). Because animal strains differ in their response to castration and subsequent androgen treatment (16, 44, 50), the effects of cadmium in the prostate of surgically castrated Wistar rats [Hsd: (WI) BR] was examined. No strain-specific differences were observed upon androgen or heavy-metal treatment (data not shown). In addition to examination of rats, the androgen-like effects of cadmium in surgically castrated mice (C57BL/6-TgN) were examined. After treatment with T, the wet weight of the prostate and seminal vesicle complex increased 4- and 3-fold, respectively. Cadmium (20 µg/kg body weight) produced a 3- and 1.5-fold increase in the wet weight of the prostate and seminal vesicle complex, respectively. The effects of the androgen and of the heavy metal were blocked by the antiandrogen CPA, suggesting a role for AR. Taken together, these results demonstrate that cadmium has androgen-like effects in target organs of castrated animals. These effects are independent of the strain and species and are mediated by the AR.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The goal of this study was to determine whether the heavy metal cadmium activates the AR. The results presented herein demonstrate that the metal has androgen-like activity in cultured cells and in castrated animals and that the effects are mediated by the AR. In LNCaP cells, the metal mimics the effects of androgens on cell growth and gene expression. The ability of an antiandrogen to block these effects suggests that the effects of cadmium are mediated through the AR. In transfection assays, the metal activates the wild-type receptor and a chimera containing the hormone-binding domain of AR. In addition, cadmium binds with high affinity to the hormone-binding domain and inhibits the binding of hormone to the receptor. The latter observation is also seen in the anterior prostate of the mouse, where cadmium blocks the binding of hormone to the AR (51). In castrated rats and mice, cadmium mimics the effects of androgens on the wet weight of the prostate gland and the seminal vesicle complex and induces the expression of an androgen-regulated gene. The in vivo effects of cadmium are also blocked by an antiandrogen, suggesting that cadmium activates AR in target tissue.

Although several earlier studies also demonstrate a mitogenic effect of low doses of cadmium in vitro and in vivo (52, 53, 54, 55), these studies did not address the mechanism of action of the metal. In human prostate epithelial cells, low concentrations of cadmium were shown to stimulate proliferation (52, 53, 54); whereas, in intact animals, addition of cadmium to the drinking water significantly increased the weight of the prostate and seminal vesicles but did not reverse the response of the gland to castration (55). In contrast to the small number of low-dose studies, numerous reports have examined the toxic and carcinogenic effects of high doses of the metal (0.3–2 mg/kg). In rodents, high doses of cadmium induce tumors at several sites, including the site of injection, lungs, testes, adrenals, the hematopoietic system, and the prostate (56, 57). The ability of cadmium to initiate prostate tumors is linked to its toxicity in the testes. Exposure of rats to doses of cadmium (approximately 0.2–0.5 mg/kg body weight), which are nontoxic to the testes, produces a dose-related increase in prostate tumors (58, 59); whereas exposure to higher doses of cadmium (>0.5 mg/kg body weight) produces testicular atrophy and reduces the number of tumors (58, 59). In addition to disrupting testicular function, high doses of cadmium also induce apoptosis in the prostate (60), adding to the complexity of the response of the gland to the metal. The ability of cadmium to activate AR, initiate transformation (16, 44, 50, 56, 61, 62), and induce apoptosis suggests that the response of the prostate is highly dependent on the concentration of the metal.

Several, but not all, epidemiological studies support a role for cadmium in the etiology of prostate cancer (6, 7, 8, 9, 10, 11, 12, 13, 50, 61, 63, 64, 65, 66, 67). The first studies to suggest a link between cadmium and prostate cancer showed an increased risk of the disease in occupationally exposed workers in a nickel-cadmium battery factory (7, 8). Several other occupational studies also demonstrate an increase in prostate cancer risk and mortality associated with cadmium exposure (9, 10). However, more recent studies have failed to show a link between occupational exposure and prostate cancer (63, 64, 65, 66). Though studies linking occupational exposure to cadmium and prostate cancer are inconsistent, studies linking environmental exposure to the metal with prostate cancer demonstrate an association between cadmium and the disease (11, 12, 13). A link between cadmium in drinking water and food and prostate cancer has also been observed (11, 13, 62, 68). It is interesting to note that the androgen-like effects of low doses of cadmium found in this study are consistent with exposure to environmental concentrations of cadmium.

Human exposure to cadmium occurs primarily through dietary sources, cigarette smoking, and occupational exposure (46, 47). The average daily intake is estimated to be 10–60 µg/d (46, 61). The metal has a biological half-life ranging from 10–30 yr (69), which may account for the significant accumulation in organs such as the prostate. Accumulation in the gland also seems to be associated with the malignant progression of prostate cancer (70, 71). In normal prostate tissue, the amount of cadmium in the nuclear fraction was 0.7 µg/g dry weight; and in benign hyperplasia, the amount of metal was 0.98 µg/g dry weight. In prostate tumors, the amount increased to 8.8 µg/g dry weight. In Nigerian black men, the amount of cadmium in the prostate gland is 8-fold higher in the cancerous tissue, compared with normal tissue (72); and in Caucasians residing in Great Britain, cadmium levels in resected prostates from patients with prostate cancer were 25 times greater than in normal prostates (73). However, no differences in cadmium concentrations between malignant and normal prostate tissue were observed in Finnish men (74). The concentrations of cadmium found in human prostate are higher than the concentrations that mimic the effects of androgens in the present study, supporting a role for the androgen-like effects of the metal in the development of prostate cancer.

Although the mechanism by which cadmium activates the AR remains to be defined, a direct (as well as an indirect) mechanism of action may be proposed. Phosphorylation, as a consequence of the activation of signal transduction pathways, has been shown to alter the activity of the AR (for review, see Ref. 75). In response to signaling pathways, the AR is phosphorylated on several serines (76), including 2 proline-directed serine phosphorylation sites in the N- terminal region (S81 and S94) and one site in the hinge region (S650) (76). Cadmium also activates protein kinase C (77, 78) and may activate AR, in part, through its effect on this pathway. However, most studies, to date, have shown that activation of protein kinase C only enhances the activity of AR in the presence of hormone (79, 80, 81). Alternatively, cadmium may activate the AR through a direct interaction with the hormone-binding domain of the receptor. Although the hormone-binding domains of nuclear receptors share less than 20% sequence homology, they have a similar structure (20, 21, 22, 23, 24, 25, 26, 27). They are composed of up to 12 {alpha}-helices (H1-H12), folded into a three-layered antiparallel helical sandwich (23, 25, 82). The central core layer usually contains three {alpha}-helices (H5/6, H9, and H10) sandwiched between 2 additional layers of helices composed of H1-4, H7, H8, and H11. The central core of the hormone-binding domain is flanked by helix H12. Upon binding, the ligand induces a conformational change, resulting in the repositioning of helix H12 over the central core (20). We have recently shown that cadmium binds to and activates ER-{alpha} through a high-affinity interaction with amino acids located on helices H4, H8, H11, and at the interface of the loop and H12 (28), suggesting that cadmium may reposition H12 in a manner similar to the repositioning observed upon ligand binding. Sequence alignment demonstrates that many of the amino acids identified as important in the interaction of cadmium with ER-{alpha} are conserved in the hormone-binding domain of the AR (26, 82). The ability of the metal to bind to and activate the hormone-binding domain of AR suggests that cadmium may activate both receptors by a similar mechanism. However, further studies are required to define the precise mechanism by which cadmium activates the AR.

Androgens play an important role in prostate cancer promotion and progression (15). Consequently, molecules that can bind to and activate the AR can potentially affect the pathogenesis of the disease. The results of this study suggest that cadmium is a candidate for such a role. At environmentally relevant concentrations, the metal mimics the effects of androgens by a mechanism that involves the hormone-binding domain of the receptor. The ability of cadmium to mimic the effects of androgens may, in part, explain the risk of prostate cancer associated with exposure to the metal.


   Acknowledgments
 
We thank Dr. Chawnshang Chang for ARA70; Dr. Robert J. Matusik for probasin; Drs. Marc E. Lippman and Anna T. Riegel for helpful discussions; and Ann Murray, Tina Wilson, Ignovie Onojafe, Aaron Foxworth, Amy Durham, Christine Johnson, Michelle Marriot, Talitha Ramey, Laura Rutter-Call, and Winsome West for technical assistance.


   Footnotes
 
This work was supported by the National Institutes of Health Grant CA-70708, American Cancer Society, and an anonymous donor. Support for tissue culture, animal, histopathology, and cell cycle analysis core facilities was provided by P50-CA-58185 and P30-CA-51008.

Abbreviations: ARA70, AR-associated protein 70; BN, nonspecific binding; BT, total binding; BS, specific binding; CAT, chloramphenicol acetyl transferase; CPA, cyproterone acetate; DHT, dihydrotestosterone; IMEM, improved MEM; Kd, dissociation constant; PSA, prostate-specific antigen; RNase, ribonuclease.

Received April 4, 2001.

Accepted for publication September 14, 2001.


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 Introduction
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 Results
 Discussion
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