Expression of Androgen Receptor Is Negatively ... - CiteSeerX

RESEARCH ARTICLE

Neoplasia . Vol. 9, No. 12, December 2007, pp. 1152 – 1159

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Expression of Androgen Receptor Is Negatively Regulated By p531 Fatouma Alimirah *, Ravichandran Panchanathan y,z,§, Jianming Chen y, Xiang Zhang §, Shuk-Mei Ho §,b and Divaker Choubey *,y,z,§,b *Edward Hines Jr. VA Hospital, Hines, IL 60141, USA; yLoyola University Chicago, Maywood, IL 60153, USA; z Cincinnati VA Medical Center, Cincinnati, OH 45220, USA; §Department of Environmental Health, University of Cincinnati, 3223 Eden Avenue, P.O. Box 670056, Cincinnati, OH 45267, USA; b University of Cincinnati Cancer Center, Cincinnati, OH, USA Abstract Increased expression of androgen receptor (AR) in prostate cancer (PC) is associated with transition to androgen independence. Because the progression of PC to advanced stages is often associated with the loss of p53 function, we tested whether the p53 could regulate the expression of AR gene. Here we report that p53 negatively regulates the expression of AR in prostate epithelial cells (PrECs). We found that in LNCaP human prostate cancer cells that express the wild-type p53 and AR and in human normal PrECs, the activation of p53 by genotoxic stress or by inhibition of p53 nuclear export downregulated the expression of AR. Furthermore, forced expression of p53 in LNCaP cells decreased the expression of AR. Conversely, knockdown of p53 expression in LNCaP cells increased the AR expression. Consistent with the negative regulation of AR expression by p53, the p53-null HCT116 cells expressed higher levels of AR compared with the isogenic HCT116 cells that express the wildtype p53. Moreover, we noted that in etoposide treated LNCaP cells p53 bound to the promoter region of the AR gene, which contains a potential p53 DNA-binding consensus sequence, in chromatin immunoprecipitation assays. Together, our observations provide support for the idea that the loss of p53 function in prostate cancer cells contributes to increased expression of AR. Neoplasia (2007) 9, 1152–1159 Keywords: Prostate cancer, androgen receptor, p53, DNA damage, transcriptional repression.

Introduction The androgen – androgen receptor (AR) signaling plays an important role in proper development and function of male reproductive organs, such as prostate and epididymis [1 – 4]. Furthermore, androgen – AR signaling plays a key role in nonreproductive organs, such as muscle, hair follicles, and brain. Abnormalities in the androgen – AR signaling pathway in humans have been linked to certain diseases, such as male infertility, Kennedy’s disease, and prostate cancer (PC) [1,2].

AR signaling is central to the development of PCs and to its response to hormone withdrawal therapy [1,4,5]. Studies have indicated that AR continues to be expressed in androgenindependent tumors and that alterations in the AR signaling contribute to the progression of PC to advanced stages, including androgen independence [1,4]. Moreover, studies have revealed that a subset of androgen-independent PCs express increased levels of AR [4,6]. However, molecular mechanisms that contribute to the increased expression of AR in PC cells remain unknown. Genetic alterations in the p53 pathway contribute to more than 50% human cancers [7 – 9]. The p53 is a transcription factor that is activated in cells in response to certain stimuli, such as DNA damage, hypoxia, oxidative stress, or other cellular stress [10 – 13]. The activation of p53 in cells results in binding of p53 to its DNA-binding consensus sequence that is present in its target genes. The binding of p53 to its target genes is known to result in either transcriptional activation of genes, such as p21CIP1 and Gadd45, or transcriptional repression of genes, such as Bcl2, MAP4, and SAK [14 – 16]. The p53-regulated genes encode proteins that mediate tumor suppressor function of p53 by inducing cell growth arrest, apoptosis, or senescence [9,15,17]. Studies have suggested that mutations in the p53 gene are associated with human PC progression [18 – 20]. Moreover, mutations in p53 may be a poor prognostic factor in PC [19,21]. Consistent with the above observations, it is interesting to note that the majority of metastatic PC – derived cell lines (seven of eight) described in the literature [22] harbor mutations of AR and/or p53. These observations suggest an important functional relationship between the p53 and AR in the progression of human PC. Because p53 and AR functionally interact with each other [23, 24], we investigated whether p53 could regulate the expression

Abbreviations: AR, androgen receptor; LMB, leptomycin-B; PC, prostate cancer; siRNA, small interfering RNA Address all correspondence to: Divaker Choubey, 3223 Eden Avenue, Kettering Complex, PO Box 670056, Cincinnati, OH 45267-0056. E-mail: [email protected] 1 This research was supported by an award from the Department of Veteran Affairs, Medical Research and Development Service (to D. C.) and CA115776 and CA112532 (to S.-M. H.). Received 20 August 2007; Revised 15 October 2007; Accepted 15 October 2007. Copyright D 2007 Neoplasia Press, Inc. All rights reserved 1522-8002/07/$25.00 DOI 10.1593/neo.07769

Regulation of AR Expression By p53

of AR. We provide evidence that p53 negatively regulates the expression of AR in human prostate epithelial cells (PrECs).

Materials and Methods Cell Lines, Culture Conditions, and Treatments Saos-2 human osteosarcoma cells stably expressing the temperature-sensitive mutant (Val138 mutation) of human p53 (with Arg-72; see Dumont et al. [25]); were generously provided by Dr. Maureen Murphy (Fox Chase Cancer Center, Philadelphia, PA). As indicated, these cells were incubated at 39jC (favoring p53 mutant conformation) or 32jC (favoring p53 wild-type conformation). HCT116 p53 wild-type and p53 knockout colorectal carcinoma cells [26] were generously provided by Dr. Bert Vogelstein (John Hopkins University Howard Hughes Medical Institute and Kimmel Cancer Center, Baltimore, MD). Saos-2 osteosarcoma and LNCaP human prostate carcinoma cell lines were purchased from the American Type Culture Collection (Manassas, VA). Saos-2 and HCT116 cell lines were maintained in RPMI-1640 and DMEM (high glucose) culture media (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum and antibiotics, respectively. LNCaP cells were maintained in 1:1 ratio of RPMI-1640 and DMEM culture media. Human normal PrECs were purchased (in culture) from Cambrex (Walkersville, MD) and maintained in prostate epithelial basal medium (PrEBM) with supplements and growth factors as suggested by the supplier. Subconfluent cultures of LNCaP cells were treated with etoposide (Calbiochem, San Diego, CA), doxorubicin (Sigma, St Louis, MO), or leptomycin-B (LMB) in ethanol (Calbiochem) at the indicated concentrations and duration. Subconfluent cultures of PrECs were treated with either doxorubicin or LMB at the indicated concentrations and duration. Knockdown of p53 Expression Subconfluent cultures of LNCaP cells were transfected with a pool of p53 small interfering RNA (siRNA) (cat # M-003557-00-05; Dharmacon, Denver, CO) or a nonspecific control siRNA (cat # D-001206-02-05; Dharmacon) as recommended by the manufacturer using Lipofectamine (Invitrogen) transfection agent and as described previously [27]. Sixty hours posttransfection, cells were processed for immunoblot analysis. Nucleofections LNCaP cells were nucleofected with 2 mg of pCMV-p53 (Val135) plasmid, encoding a temperature-sensitive mutant of human p53, or pCMV-p53 plasmid that encodes the wild type p53. Nucleofector-II device (Amaxa Biosystems, Natthermannalle 1, Germany) and nucleofection kit were used as suggested by the supplier. Twenty-four hours after nucleofection, cells were subjected to immunoblot analysis. Immunoblot Analysis and Antibodies Total cell lysates were prepared and subjected to immunoblot analysis as described previously [28]. Antibodies spe-

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cific for p53 (sc-126), p21CIP1 (sc-397), AR (cat # sc-816, sc-7305), and GAPDH (sc-32233) were purchased from Santa Cruz Biotech (Santa Cruz, CA). b-Actin antibody (cat # 4967) was purchased from Cell Signaling Technology (Danvers, MA). Horseradish peroxidase – conjugated secondary anti-mouse (NXA-931) and anti-rabbit (NA-934) antibodies were from Amersham Biosciences (Princeton, NJ). Reverse Transcription – Polymerase Chain Reaction Total RNA was extracted from Saos-2 p53 (with Arg-72; see Dumont et al. [25]) and LNCaP cells with a reagent (TRIzol; Invitrogen) and processed for cDNA synthesis followed by polymerase chain reaction (PCR) for AR and b-actin as described previously [29]. Reporter Assays Luciferase reporter assays were performed as described previously [30]. pGL-AR3.5-luc-reporter plasmid [31] was generously provided by Dr. Alexander Chlenski (Northwestern University, Chicago, IL). In brief, subconfluent cultures of Saos-2 p53 Arg 72 (Saos-2Arg72) cells were transfected with 1.8 mg of AR 3.5-luc or p21-luc [32] reporter plasmid along with 0.2 mg of pRL-TK reporter plasmid using Fugene-6 transfection reagent (Rosch, Indianapolis, IN). Twenty-four hours posttransfection, the firefly luciferase and Renilla luciferase activities were assayed (in triplicates) using Dual-Luciferase Reporter Assay kit (Promega, Madison, WI). When indicated, the transfected cells were maintained at 39jC and then shifted to 32jC. Chromatin Immunoprecipitation Assay Subconfluent cultures of LNCaP cells were treated with 45 mM of etoposide (Calbiochem) for 15 hours. The amount of 1% crosslinking mix (37% formaldehyde, 5 M NaCl, 50 mM EGTA, 1 M Hepes) was added to LNCaP cells followed by 10-minute fixation at room temperature with gentle agitation. The reaction was quenched with glycine at a final concentration of 125 mM. After centrifugation, cell pellets were washed twice with chilled 1 PBS. Cell pellets were lysed in the lysis buffer (10% SDS, 0.5 M EDTA, 1 M Tris – HCl, pH 8.0, and EDTA-free protease inhibitor cocktail; Roche, Indianapolis, IN). After brief sonication of cell lysates, extracts were subjected to immunoprecipitations. In brief, cell lysates were incubated overnight with 4 mg of anti-p53 (sc-126; Santa Cruz Biotech) or control anti-IgG (cat # OB02; Calbiochem). Immunoprecipitates were eluted with an elution buffer (1% SDS, 100 mM NaHCO3) and 500 mg/ml proteinase K and RNase A. The solution was incubated at 37jC for 30 minutes. Immunoprecipitated DNA was extracted and purified with phenol – chloroform. Purified DNA was suspended in 50 to 100 ml of sterile H2O. Polymerase chain reaction was performed at 38 cycles and the products, i.e., input DNA and immunoprecipitated DNA, were analyzed on 0.8% agarose gel. The following PCR primers were used to detect an amplification product of 1159 bp: AR (forward): 5V –CATCTGTGAAATAGAG CCTATCATATCCAG–3V; AR (backward): 5V – TAACGCCTGCCTAGTGG CTTTGGAG–3V.

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Results Expression of a Temperature-Sensitive Mutant of p53 In Human Saos-2 Cells Downregulates the Expression of AR To test whether p53 could regulate the expression of AR, we chose to use the well-characterized human osteosarcoma Saos-2 cell system (Saos-2 cells are null for p53), which expresses a temperature-sensitive mutant (Val135) of p53 with the amino acid residue Arg (instead of Pro) at the position 72 (cells indicated as SaosArg72; see Dumont et al. [25]). Moreover, Saos-2 cells express detectable levels of AR [33]. As shown in Figure 1A, incubation of SaosArg72 cells at 32jC for 24 hours resulted in upregulation of p21CIP1 protein, a transcriptional target of p53 protein. Importantly, levels of AR protein decreased measurably in extracts from SaosArg72 cells that were incubated at 32jC. To rule out the possibility that incubation of cells at 32jC could account for decreases in the AR protein levels (independent of p53 expression), we also compared AR protein levels between the parental Saos-2 cells that were incubated at 39 or 32jC. We found no measurable difference between AR protein levels in extracts from cells incubated at these two different temperatures (Figure 1B). We also noted that the steady-state levels of AR mRNA also decreased about more than two-fold in SaosArg72 cells after their incubation at 32jC for 24 hours (Figure 1C). Consistent with the above observations that incubation of SaosArg72 cells at 32jC resulted in upregulation of p21CIP1

expression and downregulation of AR expression, we noted that incubation of SaosArg72 cells that were transfected with p21-luc-reporter plasmid at 32jC resulted in stimulation of the activity of reporter about eight-fold (Figure 1D). In contrast, incubation of SaosArg72 cells that were transfected with AR3.5-luc-reporter plasmid at 32jC resulted in f50% decrease in the activity of reporter in two experiments (Figure 1E ). Together, these observations suggested that the restoration of p53 function in Saos-2 cells downregulated the AR expression. The p53 Expression Status In Isogenic HCT116 Cell Lines Inversely Correlates with the Expression Levels of AR Our above observations that the functional activation of a temperature-sensitive mutant of p53 in SaosArg72 cells downregulated the expression of AR prompted us to compare the AR protein levels between human colorectal cancer cell line HCT116 cells (HCT116p53+) that express the wild type p53 and isogenic HCT116 cells (HCT116p53) that are null for p53 expression [26]. Moreover, HCT116 cells express detectable levels of AR [34]. As shown in Figure 2, we could detect the expression of p53 in the HCT116p53+ cells, but not in HCT116p53 cells (compare lane 1 with 2). Notably, consistent with p53 expression status in these two human cancer cell lines, we could detect the expression of p21CIP1 in HCT116p53+ cells, but not in HCT116p53 cells (compare lane 1 with 2). Importantly, the expression of p53 status in these two isogenic cell lines inversely correlated with the

Figure 1. Restoration of p53 function in human Saos-2 osteosarcoma cell line downregulates the expression of AR. (A) Subconfluent cultures of Saos Arg72 cells were incubated either at 39jC (lanes 1) or at 32jC (lane 2) for 24 hours. After incubations, total cell lysates were analyzed by immunoblot analysis using antibodies specific to the indicated proteins. (B) Subconfluent cultures of Saos-2 cells were incubated either at 39jC (lanes 1) or at 32jC (lane 2) for 24 hours. After incubations, total cell lysates were analyzed by immunoblot analysis using antibodies specific to the indicated proteins. (C) Subconfluent cultures of Saos Arg72 cells were incubated either at 39jC (lanes 1) or 32jC (lanes 2). Twenty-four hours after incubation, total RNA was isolated and steady-state levels of AR and actin mRNA were analyzed by semiquantitative reverse transcription – polymerase chain reaction (RT-PCR). (D and E) Two sets of Saos Arg72 cell cultures (in 60-mm plates) were transfected with p21-luc or AR3.5-luc-reporter plasmid (1.8 g) along with pRL-TK plasmid (0.2 g; plasmids in 9:1 ratio) using Fugene-6 transfection reagent. One set of plates for each reporter was incubated at 39jC and the other sets of plates were incubated at 32jC. Forty-four hours after incubations, cells were processed for dual-luciferase reporter activity assays as described in the Materials and Methods section. The firefly luciferase reporter activity was normalized to the Renilla luciferase activity to control for variations in transfection efficiencies. The luciferase activity for (C) p21-luc or (D) AR3.5-luc reporter in control cells is shown as 1.



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(pCMV) at 32jC for 24 hours did not result in the downregulation of AR protein levels (data not shown). Similarly, nucleofection of LNCaP cells with pCMV-p53 plasmid, but not an empty pCMV vector, also resulted in the downregulation of AR protein levels (Figure 3B).

Figure 2. The p53 status in isogenic HCT116 cell lines inversely correlates with the expression levels of AR. Total cell extracts prepared from subconfluent cultures of HCT116 p53+/+ (lane 1) or HCT116 p53/ (lane 2) cells were analyzed by immunoblot analysis using antibodies specific to the indicated proteins.

expression levels of AR. These observations suggested that the expression of functional p53 in HCT116 cells is associated with reduced or lack of AR expression. Expression of a Temperature-Sensitive Mutant of p53 In LNCaP Cells Downregulates AR Expression Our above observations that the expression of the functional p53 in two human cancer cell lines (Saos-2 and HCT116) inversely correlated with the expression levels of AR prompted us to test whether the expression of a temperaturesensitive mutant of p53 or the ectopic expression of p53 in LNCaP PC cells that are known to express relatively high levels of a mutant AR and detectable levels of functional p53 downregulates the AR expression. As shown in Figure 3A, incubation of LNCaP cells that were nucleofected with a temperature-sensitive (Val135) mutant of p53 at 32jC for 24 hours resulted in upregulation of p21CIP1 protein levels. More importantly, the incubation of cells downregulated the endogenous levels of AR protein. However, the incubation of LNCaP cells that were nucleofected with an empty vector

Knockdown of p53 Expression In LNCaP Cells Upregulates AR Expression Our above observations that the expression of a temperature-sensitive mutant of p53 or the ectopic expression of p53 in LNCaP PC cells downregulated the expression of AR prompted us to test whether knockdown of p53 in LNCaP cells upregulates the expression of AR. As shown in Figure 3C, knockdown of p53 in LNCaP with siRNA resulted in >70% decreases in p53 protein levels (compare lane 2 with 1). Moreover, the knockdown resulted in >70% decreases in p21CIP1 protein levels and f50% increases in Bcl2 levels, a p53-repressible gene [35]. Importantly, the knockdown of p53 resulted in f2.5- to 3-fold increases in AR protein levels. These observations suggested that the steadystate levels of AR in LNCaP cells are regulated by p53. Activation of p53 In LNCaP Cells By Doxorubicin, Etoposide, or LMB Downregulates the AR Expression Treatment of LNCaP cells with DNA-damaging agents, such as doxorubicin [36] or etoposide [37], is known to activate p53. Moreover, treatment of LNCaP with LMB, an inhibitor of nuclear export, is also known to activate p53 [38]. Therefore, we tested whether the activation of p53 in LNCaP cells by DNA-damaging agents or by LMB treatment downregulates the expression of AR. As shown in Figure 4A, treatment of cells with doxorubicin at the indicated concentration for 18 hours resulted in stabilization of p53, which correlated well with upregulation of p21CIP1 protein levels. Of note, upregulation of p53 levels after doxorubicin treatment was associated with downregulation of AR protein levels.

Figure 3. Expression levels of p53 in LNCaP cells determine the expression levels of AR. (A) LNCaP cells were nucleofected with pCMV-p53 (Val138) plasmid as described in the Materials and Methods section. One set of nucleofected cells was incubated at 39jC (lane 1) and the other sets of cells were incubated at 32jC (lane 2) for 24 hours. Cells were harvested after 24 hours and total cell extracts were analyzed by immunoblot analysis using antibodies specific to the indicated proteins. (B) LNCaP cells were nucleofected with pCMV (lane 1) or pCMV-p53 (lane 2) plasmid as described in the Materials and Methods section. Nucleofected cells were incubated at 37jC for 24 hours. Total cell extracts were analyzed by immunoblot analysis using antibodies specific to the indicated proteins. (C) LNCaP cells were transfected with either control siRNA (lane 1) or a pool of p53 siRNA (lane 2) as described in the Materials and Methods section using Lipofectamine transfection reagent. Sixty hours after transfection of cells, total cell lysates were analyzed by immunoblot analysis using antibodies specific to the indicated proteins.


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Figure 4. Treatment of LNCaP cells with doxorubicin, etoposide, or LMB downregulates the expression of AR. (A) Subconfluent cultures of LNCaP cells were treated with the indicated amounts (g/ml) of doxorubicin for 18 hours. After the treatment, cell lysates were analyzed by immunoblot analysis using antibodies specific to the indicated proteins. (B) Subconfluent cultures of LNCaP cells were treated with the indicated concentrations (M) of etoposide for 15 hours. After the treatment, cell lysates were analyzed by immunoblot analysis using antibodies specific to the indicated proteins. (C) Subconfluent cultures of LNCaP cells were treated with the indicated concentrations (nM) of LMB for 24 hours. After the treatment, cell lysates were analyzed by immunoblot analysis using antibodies specific to the indicated proteins. (D) Subconfluent cultures of LNCaP cells were treated 5 (lane 2), 10 (lane 3), or 20 nM concentrations of LMB for 24 hours. As a control, cells were left untreated (lane 1). After the treatment, total RNA was isolated and the steady state levels AR and actin mRNA were analyzed by semiquantitative RT-PCR.

Similarly, treatment of cells with the indicated concentration of etoposide for 15 hours, which resulted in upregulation of p53 and p21CIP1, was associated with downregulation of AR protein levels. Moreover, treatment of cells with LMB at the indicated concentration for 24 hours resulted in increases in p53 protein levels that were associated with upregulation of p21CIP1 protein levels. Interestingly, increases in p53 protein levels were associated with downregulation of AR protein (Figure 4C) and mRNA (Figure 4D) levels. Together, these observations suggested that the activation of p53 in LNCaP cells by treatment of cells by doxorubicin, etoposide, or LMB resulted in downregulation of AR expression. Activation of p53 In Normal Human PrECs By Doxorubicin and LMB Results in Decreases in AR Levels The above observations that the activation of p53 in LNCaP cells by treatment of cells by doxorubicin, etoposide, or LMB resulted in downregulation of AR expression, prompted us to test whether the activation of p53 in normal human PrECs also results in decreases in AR protein levels. As shown in Figure 5A, treatment of PrECs with doxorubicin at the indicated increasing concentration for 19 hours resulted in accumulation of p53, which correlated with increases in p21CIP1 protein levels. Of note, increases in p53 protein levels were associated with decreases in AR protein levels. Similarly, the treatment of normal PrECs with LMB for 16 hours resulted in an increase in p53 and p21CIP1 protein levels (compare lane 2 with 1). However, the increase in p53 protein levels

was associated with the decreases in AR protein levels. Together, these observations suggested that the activation of p53 in normal PrECs also results in downregulation of AR protein levels. p53 Associates with the 5 V-Regulatory Region of the AR Gene In LNCaP Cells After Etoposide Treatment Our above observations that the activation of p53 function in Saos-2 (Figure 1B) and LNCaP (Figure 4D) cells

Figure 5. Treatment of normal human prostate epithelial cells with doxorubicin or LMB downregulates AR expression. (A) Subconfluent cultures of normal PrECs were treated with the indicated amounts (g/ml) of doxorubicin for 19 hours. After the treatment, cell lysates were analyzed by immunoblot analysis using antibodies specific to the indicated proteins. (B) Subconfluent cultures of normal PrECs were treated with the indicated concentration (nM) of LMB for 16 hours. After the treatment, cell lysates were analyzed by immunoblot analysis using antibodies specific to the indicated proteins.



decreased the steady-state levels of AR mRNA and in Saos-2 cells repressed the activity of AR3.5-luc reporter prompted us to search for a potential p53 DNA – binding site consensus sequence in the 5V-regulatory region of AR gene. Our search identified a single potential p53 DNA – binding site (nucleotides 5290 – 5309; GenBank accession number X78592) in the 5V-regulatory region of the human AR gene (Figure 6A). This potential p53 DNA – binding site is located 469 bp upstream to the mRNA start site in the promoter region of the human AR gene (Figure 6B). After localization of a potential p53 DNA –binding site in the promoter region of AR gene, we sought to determine whether p53 associates with the 5V-regulatory region of AR gene. As shown in Figure 6C, we were unable to detect any association of p53 protein in control untreated LNCaP cells with the 5V-regulatory region of AR gene in chromatin immunoprecipitation assays. However, treatment of LNCaP cells with etoposide, which upregulated p53 protein levels (Figure 4B), resulted in an association of p53 protein with the regulatory region (compare lane 7 with 6) of the AR gene. Together, these observations indicated that the activation of p53 in LNCaP cells with etoposide, which resulted in downregulation of AR expression, was associated with the binding of p53 protein to the 5V-regulatory region of the AR gene. Together, these observations provide support to the idea that p53 negatively regulates the transcription of AR gene in LNCaP cells.

Figure 6. p53 associates with the 5V-regulatory region of the AR gene. (A) A comparison of the p53 DNA – binding site in the human AR gene with known p53 DNA – binding sites in other genes. A bold nucleotide indicates a variation from the p53 DNA – binding consensus sequence. (B) The 5V-regulatory region of human AR gene indicating the locations of the potential p53 DNA – binding site (nucleotides 5290 – 5309), two primers that were used for PCR (after chromatin precipitation), and the mRNA start site (nucleotide 5778) in the AR gene (using the reference GenBank accession no. X78592). (C) Chromatin was prepared from LNCaP cells either left untreated or treated with etoposide (45 M for 15 hours). Chromatin was incubated with antibodies to p53 (lanes 6 and 7) or, as a negative control, with an isotype antibody (lanes 4 and 5). DNA was extracted from immunoprecipitates and PCR-amplified using a pair of primers that flanked the p53 DNA – binding site in the promoter region of the AR gene. As a positive control, we also amplified the input chromatin DNA from control (lane 2) or etoposide- (lane 3) treated cells. As a negative control, we did not add any DNA in the PCR (lane 1).


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Discussion Expression of AR in PrECs contributes to increased cell proliferation and survival [39,40]. Because androgen signaling through AR activates transcription of genes that mediate the cell growth regulatory functions of the AR [2,3,41,42], it is important to understand the molecular mechanisms that regulate expression of the AR gene and the activity of AR. Regulation of AR activity in PrECs can be achieved in several different ways [3,4]: modulation of AR gene expression, androgen binding to AR, AR nuclear translocation, AR protein stability, and AR transactivation. Importantly, studies have provided evidence that a modest increase in AR mRNA was the only change consistently associated with the development of resistance to antiandrogen therapy [4,6]. Moreover, this increase in AR mRNA levels and protein was both necessary and sufficient to convert PC growth from a hormone-sensitive to a hormone-refractory stage [6]. Furthermore, a study suggested that the expression of gain-of-function mutants of p53 in LNCaP cells results in androgen-independent cell growth [20]. Because mutations in the p53 gene are associated with the progression of PC to advance stages [18–20], our observations provide support for the idea that mutations in p53 gene in PC cells contribute to increased expression of AR and the progression to androgen independence. It has been noted that male p53-null mice have less apoptosis in the prostate glands that is associated with the first 4 days after castration compared to wild-type mice [43]. Moreover, expression of the transgene SV40 large T-antigen in PrECs is known to result in adenocarcinoma of the mouse prostate [44]. Because SV40 large T-antigen binds to p53 and inactivates it [7], it is conceivable that inactivation of p53 function by the large T-antigen in mouse PrECs, in part, contributes to the upregulation of AR expression and the development of adenocarcinoma of the prostate. Therefore, further work will be needed to test this hypothesis. Treatment of LNCaP cells with murine double minute-2 antagonist nutlin-3 downregulates the AR expression and inhibits AR recruitment to promoters of the AR-responsive genes [45]. Because nutlin-3 treatment of cells results in increased levels of p53, our observations raise the possibility that nutlin-3 – mediated increased levels of p53 downregulate the expression of AR, resulting in inhibition of recruitment of AR to the promoters of AR-responsive genes. Knockdown of p53 expression in LNCaP cells results in upregulation of AR expression [46]. Therefore, our observations (Figure 3B) that knockdown of p53 in LNCaP cells resulted in downregulation of p21CIP1, and upregulation of Bcl2 and AR are consistent with the above observations. Moreover, we noted that levels of AR were detectable in HCT116p53/ cells, but not in isogenic HCT116p53+/+ cells. Together, these observations support the idea that p53 negatively regulates the expression of AR in LNCaP and HCT116 cells. Previous studies have revealed that increased levels of endogenous as well as exogenous p53 in PC cells lead to growth inhibition and apoptosis in vitro and in vivo [19, 47 – 49]. Because expression of AR is important for the proliferation and survival of PrECs [39,40] and increased expression of AR is associated with androgen independence

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[6], our observations provide support to the idea that the expression of functional p53 in normal PrECs and in PC cells by negatively regulating the expression of AR also regulates cell proliferation and survival [39,40]. Consistent with this idea, we noted that forced expression of functional p53 in LNCaP cells reduced cell proliferation (data not shown). Treatment of LNCaP cells with high concentrations of dihydrotestosterone (10 mM) downregulates the p53 mRNA levels, whereas androgen deprivation of cells results in reduced levels of p53 protein [46]. Therefore, our observations that increased levels of p53 in LNCaP cells negatively regulate the expression levels of AR mRNA and protein suggest that there is mutual regulation of expression between p53 and AR. Transcriptional activation by p53 requires the interaction of the protein as a tetramer with a consensus binding site consisting of two half sites, each comprising two copies of the sequence PuPuPuC(A/T) arranged head-to-head and separated by 0 to 13 bp [7]. In addition to the transcriptional activation, p53 has been shown to repress the transcription of certain genes [16,32,50]. Moreover, p53 is shown to repress the transcription of genes in human PC cells [51]. However, the molecular mechanisms for p53-mediated transcriptional repression are complex and appear to depend on the orientation of the p53 DNA–binding sequence (for example, head-to-head vs head-to-tail orientation) and the promoter context [15]. Interestingly, we report the presence of a potential p53 DNA– binding site (a head-to-head site) in the promoter region of the human AR gene. Moreover, we could detect binding of p53 to the promoter region of the AR gene in chromatin precipitation assays after treatment of LNCaP cells with etoposide that resulted in stabilization of p53 and downregulation of AR levels. However, we could not detect the binding of p53 to the promoter region in untreated LNCaP cells. Therefore, our above observations make it likely that the increased levels of p53 in LNCaP cells negatively regulate the transcription of AR gene through binding to a head-to-head p53 DNA –binding site in the promoter region. Because molecular mechanisms for transcriptional repression by p53 are relatively very complex, further studies will be needed to determine how p53 represses the transcription of human AR gene. In summary, our observations will serve basis to understand the molecular mechanisms that contribute to increased expression of AR in advanced human PCs.

Acknowledgements We thank M. Murphy and B. Vogelstein for generously providing cell lines and A. Chlenski for generously providing the AR-reporter plasmids.

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