Expression of Androgen Receptor Is Negatively Regulated by P531

RESEARCH ARTICLE Neoplasia . Vol. 9, No. 12, December 2007, pp. 1152 – 1159 1152 www.neoplasia.com

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; zCincinnati 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; bUniversity of Cincinnati Cancer Center, Cincinnati, OH, USA

Abstract Increased expression of androgen receptor (AR) in AR signaling is central to the development of PCs and to its prostate cancer (PC) is associated with transition to response to hormone withdrawal therapy [1,4,5]. Studies have androgen independence. Because the progression of indicated that AR continues to be expressed in androgen- PC to advanced stages is often associated with the independent tumors and that alterations in the AR signaling loss of p53 function, we tested whether the p53 could contribute to the progression of PC to advanced stages, includ- regulate the expression of AR gene. Here we report ing androgen independence [1,4]. Moreover, studies have that p53 negatively regulates the expression of AR revealed that a subset of androgen-independent PCs express in prostate epithelial cells (PrECs). We found that in increased levels of AR [4,6]. However, molecular mechanisms LNCaP human prostate cancer cells that express the that contribute to the increased expression of AR in PC cells wild-type p53 and AR and in human normal PrECs, remain unknown. the activation of p53 by genotoxic stress or by inhibi- Genetic alterations in the p53 pathway contribute to more tion of p53 nuclear export downregulated the expres- than 50% human cancers [7–9]. The p53 is a transcription sion of AR. Furthermore, forced expression of p53 in factor that is activated in cells in response to certain stimuli, LNCaP cells decreased the expression of AR. Con- such as DNA damage, hypoxia, oxidative stress, or other versely, knockdown of p53 expression in LNCaP cells cellular stress [10–13]. The activation of p53 in cells results increased the AR expression. Consistent with the nega- in binding of p53 to its DNA-binding consensus sequence that tive regulation of AR expression by p53, the p53-null is present in its target genes. The binding of p53 to its target HCT116 cells expressed higher levels of AR compared genes is known to result in either transcriptional activation of with the isogenic HCT116 cells that express the wild- genes, such as p21CIP1 and Gadd45, or transcriptional repres- type p53. Moreover, we noted that in etoposide treated sion of genes, such as Bcl2, MAP4, and SAK [14–16]. The LNCaP cells p53 bound to the promoter region of the p53-regulated genes encode proteins that mediate tumor sup- AR gene, which contains a potential p53 DNA-binding pressor function of p53 by inducing cell growth arrest, apop- consensus sequence, in chromatin immunoprecipitation tosis, or senescence [9,15,17]. assays. Together, our observations provide support for Studies have suggested that mutations in the p53 gene are the idea that the loss of p53 function in prostate cancer associated with human PC progression [18–20]. Moreover, cells contributes to increased expression of AR. mutations in p53 may be a poor prognostic factor in PC [19,21]. Neoplasia (2007) 9, 1152–1159 Consistent with the above observations, it is interesting to note that the majority of metastatic PC–derived cell lines (seven of Keywords: Prostate cancer, androgen receptor, p53, DNA damage, transcriptional repression. 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, Introduction 24], we investigated whether p53 could regulate the expression The androgen–androgen receptor (AR) signaling plays an important role in proper development and function of male reproductive organs, such as prostate and epididymis Abbreviations: AR, androgen receptor; LMB, leptomycin-B; PC, prostate cancer; siRNA, small [1–4]. Furthermore, androgen–AR signaling plays a key interfering RNA Address all correspondence to: Divaker Choubey, 3223 Eden Avenue, Kettering Complex, PO role in nonreproductive organs, such as muscle, hair fol- Box 670056, Cincinnati, OH 45267-0056. E-mail: [email protected] 1 licles, and brain. Abnormalities in the androgen–AR sig- 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.). naling pathway in humans have been linked to certain Received 20 August 2007; Revised 15 October 2007; Accepted 15 October 2007. diseases, such as male infertility, Kennedy’s disease, and Copyright D 2007 Neoplasia Press, Inc. All rights reserved 1522-8002/07/$25.00 prostate cancer (PC) [1,2]. DOI 10.1593/neo.07769 Regulation of AR Expression By p53 Alimirah et al. 1153 of AR. We provide evidence that p53 negatively regulates the cific for p53 (sc-126), p21CIP1 (sc-397), AR (cat # sc-816, expression of AR in human prostate epithelial cells (PrECs). 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 Materials and Methods (Danvers, MA). Horseradish peroxidase–conjugated sec- ondary anti-mouse (NXA-931) and anti-rabbit (NA-934) anti- Cell Lines, Culture Conditions, and Treatments bodies were from Amersham Biosciences (Princeton, NJ). Saos-2 human osteosarcoma cells stably expressing the temperature-sensitive mutant (Val138 mutation) of human Reverse Transcription–Polymerase Chain Reaction p53 (with Arg-72; see Dumont et al. [25]); were generously Total RNA was extracted from Saos-2 p53 (with Arg-72; provided by Dr. Maureen Murphy (Fox Chase Cancer Center, see Dumont et al. [25]) and LNCaP cells with a reagent Philadelphia, PA). As indicated, these cells were incubated (TRIzol; Invitrogen) and processed for cDNA synthesis fol- at 39jC (favoring p53 mutant conformation) or 32jC (favor- lowed by polymerase chain reaction (PCR) for AR and ing p53 wild-type conformation). b-actin as described previously [29]. HCT116 p53 wild-type and p53 knockout colorectal carcinoma cells [26] were generously provided by Dr. Bert Vogelstein (John Hopkins University Howard Hughes Medi- Reporter Assays cal Institute and Kimmel Cancer Center, Baltimore, MD). Luciferase reporter assays were performed as described Saos-2 osteosarcoma and LNCaP human prostate carcinoma previously [30]. pGL-AR3.5-luc-reporter plasmid [31] was cell lines were purchased from the American Type Culture generously provided by Dr. Alexander Chlenski (Northwestern Collection (Manassas, VA). Saos-2 and HCT116 cell lines University, Chicago, IL). In brief, subconfluent cultures of Arg72 were maintained in RPMI-1640 and DMEM (high glucose) Saos-2 p53 Arg 72 (Saos-2 ) cells were transfected with culture media (Invitrogen, Carlsbad, CA) supplemented with 1.8 mg of AR 3.5-luc or p21-luc [32] reporter plasmid along 10% (v/v) fetal bovine serum and antibiotics, respectively. with 0.2 mg of pRL-TK reporter plasmid using Fugene-6 trans- LNCaP cells were maintained in 1:1 ratio of RPMI-1640 and fection reagent (Rosch, Indianapolis, IN). Twenty-four hours DMEM culture media. Human normal PrECs were purchased posttransfection, the firefly luciferase and Renilla luciferase (in culture) from Cambrex (Walkersville, MD) and maintained activities were assayed (in triplicates) using Dual-Luciferase in prostate epithelial basal medium (PrEBM) with supple- Reporter Assay kit (Promega, Madison, WI). When indicated, ments and growth factors as suggested by the supplier. Sub- the transfected cells were maintained at 39jC and then shifted confluent cultures of LNCaP cells were treated with etoposide to 32jC. (Calbiochem, San Diego, CA), doxorubicin (Sigma, St Louis, MO), or leptomycin-B (LMB) in ethanol (Calbiochem) at the Chromatin Immunoprecipitation Assay indicated concentrations and duration. Subconfluent cultures Subconfluent cultures of LNCaP cells were treated with of PrECs were treated with either doxorubicin or LMB at the 45 mM of etoposide (Calbiochem) for 15 hours. The amount indicated concentrations and duration. of 1% crosslinking mix (37% formaldehyde, 5 M NaCl, 50 mM EGTA, 1 M Hepes) was added to LNCaP cells followed by Knockdown of p53 Expression 10-minute fixation at room temperature with gentle agitation. Subconfluent cultures of LNCaP cells were transfected The reaction was quenched with glycine at a final concen- with a pool of p53 small interfering RNA (siRNA) (cat # tration of 125 mM. After centrifugation, cell pellets were M-003557-00-05; Dharmacon, Denver, CO) or a nonspecific washed twice with chilled 1 PBS. Cell pellets were lysed control siRNA (cat # D-001206-02-05; Dharmacon) as rec- in the lysis buffer (10% SDS, 0.5 M EDTA, 1 M Tris–HCl, pH ommended by the manufacturer using Lipofectamine (Invitro- 8.0, and EDTA-free protease inhibitor cocktail; Roche, India- gen) transfection agent and as described previously [27]. Sixty napolis, IN). After brief sonication of cell lysates, extracts hours posttransfection, cells were processed for immunoblot were subjected to immunoprecipitations. In brief, cell lysates analysis. were incubated overnight with 4 mg of anti-p53 (sc-126; Santa Cruz Biotech) or control anti-IgG (cat # OB02; Calbio- Nucleofections chem). Immunoprecipitates were eluted with an elution buffer LNCaP cells were nucleofected with 2 mg of pCMV-p53 (1% SDS, 100 mM NaHCO3) and 500 mg/ml proteinase K (Val135) plasmid, encoding a temperature-sensitive mutant and RNase A. The solution was incubated at 37jCfor of human p53, or pCMV-p53 plasmid that encodes the 30 minutes. Immunoprecipitated DNA was extracted and puri- wild type p53. Nucleofector-II device (Amaxa Biosystems, fied with phenol–chloroform. Purified DNA was suspended Natthermannalle 1, Germany) and nucleofection kit were in 50 to 100 ml of sterile H2O. Polymerase chain reaction was used as suggested by the supplier. Twenty-four hours after performed at 38 cycles and the products, i.e., input DNA and nucleofection, cells were subjected to immunoblot analysis. immunoprecipitated DNA, were analyzed on 0.8% agarose gel. The following PCR primers were used to detect an Immunoblot Analysis and Antibodies amplification product of 1159 bp: AR (forward): 5V–CATCTGT- Total cell lysates were prepared and subjected to immu- GAAATAGAG CCTATCATATCCAG–3V; AR (backward): 5V– noblot analysis as described previously [28]. Antibodies spe- TAACGCCTGCCTAGTGG CTTTGGAG–3V.

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Results expression and downregulation of AR expression, we noted that incubation of SaosArg72 cells that were transfected with Expression of a Temperature-Sensitive Mutant of p21-luc-reporter plasmid at 32jC resulted in stimulation of p53 In Human Saos-2 Cells Downregulates the the activity of reporter about eight-fold (Figure 1D). In con- Expression of AR trast, incubation of SaosArg72 cells that were transfected To test whether p53 could regulate the expression of AR, with AR3.5-luc-reporter plasmid at 32jC resulted in f50% we chose to use the well-characterized human osteosar- decrease in the activity of reporter in two experiments (Fig- coma Saos-2 cell system (Saos-2 cells are null for p53), ure 1E ). Together, these observations suggested that the which expresses a temperature-sensitive mutant (Val135) of restoration of p53 function in Saos-2 cells downregulated p53 with the amino acid residue Arg (instead of Pro) at the the AR expression. position 72 (cells indicated as SaosArg72; see Dumont et al. [25]). Moreover, Saos-2 cells express detectable levels of AR The p53 Expression Status In Isogenic HCT116 Cell [33]. As shown in Figure 1A, incubation of SaosArg72 cells at Lines Inversely Correlates with the Expression 32jC for 24 hours resulted in upregulation of p21CIP1 protein, Levels of AR a transcriptional target of p53 protein. Importantly, levels of Our above observations that the functional activation of a AR protein decreased measurably in extracts from SaosArg72 temperature-sensitive mutant of p53 in SaosArg72 cells down- cells that were incubated at 32jC. regulated the expression of AR prompted us to compare the To rule out the possibility that incubation of cells at 32jC AR protein levels between human colorectal cancer cell line could account for decreases in the AR protein levels (inde- HCT116 cells (HCT116p53+) that express the wild type p53 pendent of p53 expression), we also compared AR protein and isogenic HCT116 cells (HCT116p53) that are null for p53 levels between the parental Saos-2 cells that were incubated expression [26]. Moreover, HCT116 cells express detectable at 39 or 32jC. We found no measurable difference between levels of AR [34]. As shown in Figure 2, we could detect AR protein levels in extracts from cells incubated at these the expression of p53 in the HCT116p53+ cells, but not in two different temperatures (Figure 1B). HCT116p53 cells (compare lane 1 with 2). Notably, con- We also noted that the steady-state levels of AR mRNA sistent with p53 expression status in these two human can- also decreased about more than two-fold in SaosArg72 cells cer cell lines, we could detect the expression of p21CIP1 in after their incubation at 32jC for 24 hours (Figure 1C). HCT116p53+ cells, but not in HCT116p53 cells (compare Consistent with the above observations that incubation of lane 1 with 2). Importantly, the expression of p53 status in SaosArg72 cells at 32jC resulted in upregulation of p21CIP1 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 SaosArg72 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 SaosArg72 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 SaosArg72 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.

Neoplasia . Vol. 9, No. 12, 2007 Regulation of AR Expression By p53 Alimirah et al. 1155

(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).

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

Figure 2. The p53 status in isogenic HCT116 cell lines inversely correlates with of AR prompted us to test whether knockdown of p53 in the expression levels of AR. Total cell extracts prepared from subconfluent LNCaP cells upregulates the expression of AR. As shown in p53+/+ p53/ cultures of HCT116 (lane 1) or HCT116 (lane 2) cells were analyzed Figure 3C, knockdown of p53 in LNCaP with siRNA resulted by immunoblot analysis using antibodies specific to the indicated proteins. in >70% decreases in p53 protein levels (compare lane 2 with 1). Moreover, the knockdown resulted in >70% de- expression levels of AR. These observations suggested that creases in p21CIP1 protein levels and f50% increases in the expression of functional p53 in HCT116 cells is associ- Bcl2 levels, a p53-repressible gene [35]. Importantly, the ated with reduced or lack of AR expression. knockdown of p53 resulted in f2.5- to 3-fold increases in AR protein levels. These observations suggested that the steady- Expression of a Temperature-Sensitive Mutant of p53 state levels of AR in LNCaP cells are regulated by p53. In LNCaP Cells Downregulates AR Expression Our above observations that the expression of the func- Activation of p53 In LNCaP Cells By Doxorubicin, tional p53 in two human cancer cell lines (Saos-2 and Etoposide, or LMB Downregulates the AR Expression HCT116) inversely correlated with the expression levels of AR Treatment of LNCaP cells with DNA-damaging agents, prompted us to test whether the expression of a temperature- such as doxorubicin [36] or etoposide [37], is known to ac- sensitive mutant of p53 or the ectopic expression of p53 in tivate p53. Moreover, treatment of LNCaP with LMB, an in- LNCaP PC cells that are known to express relatively high hibitor of nuclear export, is also known to activate p53 [38]. levels of a mutant AR and detectable levels of functional p53 Therefore, we tested whether the activation of p53 in LNCaP downregulates the AR expression. As shown in Figure 3A, cells by DNA-damaging agents or by LMB treatment down- incubation of LNCaP cells that were nucleofected with a regulates the expression of AR. As shown in Figure 4A, temperature-sensitive (Val135) mutant of p53 at 32jC for 24 treatment of cells with doxorubicin at the indicated concen- hours resulted in upregulation of p21CIP1 protein levels. More tration for 18 hours resulted in stabilization of p53, which importantly, the incubation of cells downregulated the en- correlated well with upregulation of p21CIP1 protein levels. Of dogenous levels of AR protein. However, the incubation of note, upregulation of p53 levels after doxorubicin treatment LNCaP cells that were nucleofected with an empty vector 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 was associated with the decreases in AR protein levels. To- of etoposide for 15 hours, which resulted in upregulation of gether, these observations suggested that the activation of p53 and p21CIP1, was associated with downregulation of AR p53 in normal PrECs also results in downregulation of AR protein levels. Moreover, treatment of cells with LMB at the protein levels. indicated concentration for 24 hours resulted in increases in p53 protein levels that were associated with upregulation of p53 Associates with the 5V-Regulatory Region of the AR p21CIP1 protein levels. Interestingly, increases in p53 protein Gene In LNCaP Cells After Etoposide Treatment levels were associated with downregulation of AR protein Our above observations that the activation of p53 func- (Figure 4C) and mRNA (Figure 4D) levels. Together, these tion in Saos-2 (Figure 1B) and LNCaP (Figure 4D) cells 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 accu- mulation of p53, which correlated with increases in p21CIP1 Figure 5. Treatment of normal human prostate epithelial cells with doxorubicin or LMB downregulates AR expression. (A) Subconfluent cultures of normal protein levels. Of note, increases in p53 protein levels were PrECs were treated with the indicated amounts (g/ml) of doxorubicin for associated with decreases in AR protein levels. Similarly, 19 hours. After the treatment, cell lysates were analyzed by immunoblot the treatment of normal PrECs with LMB for 16 hours resulted analysis using antibodies specific to the indicated proteins. (B) Subconfluent CIP1 cultures of normal PrECs were treated with the indicated concentration (nM) of in an increase in p53 and p21 protein levels (compare LMB for 16 hours. After the treatment, cell lysates were analyzed by immuno- lane 2 with 1). However, the increase in p53 protein levels blot analysis using antibodies specific to the indicated proteins.

Neoplasia . Vol. 9, No. 12, 2007 Regulation of AR Expression By p53 Alimirah et al. 1157 decreased the steady-state levels of AR mRNA and in Discussion Saos-2 cells repressed the activity of AR3.5-luc reporter Expression of AR in PrECs contributes to increased cell pro- prompted us to search for a potential p53 DNA–binding site liferation and survival [39,40]. Because androgen signaling consensus sequence in the 5V-regulatory region of AR gene. through AR activates transcription of genes that mediate the Our search identified a single potential p53 DNA–binding cell growth regulatory functions of the AR [2,3,41,42], it is im- site (nucleotides 5290–5309; GenBank accession number portant to understand the molecular mechanisms that regu- X78592) in the 5V-regulatory region of the human AR gene late expression of the AR gene and the activity of AR. (Figure 6A). This potential p53 DNA–binding site is located Regulation of AR activity in PrECs can be achieved in sev- 469 bp upstream to the mRNA start site in the promoter eral different ways [3,4]: modulation of AR gene expression, region of the human AR gene (Figure 6B). After localization androgen binding to AR, AR nuclear translocation, AR protein of a potential p53 DNA–binding site in the promoter region stability, and AR transactivation. Importantly, studies have of AR gene, we sought to determine whether p53 associates provided evidence that a modest increase in AR mRNA was with the 5V-regulatory region of AR gene. As shown in Fig- the only change consistently associated with the development ure 6C, we were unable to detect any association of p53 of resistance to antiandrogen therapy [4,6]. Moreover, this in- protein in control untreated LNCaP cells with the 5V-regulatory crease in AR mRNA levels and protein was both necessary region of AR gene in chromatin immunoprecipitation assays. and sufficient to convert PC growth from a hormone-sensitive However, treatment of LNCaP cells with etoposide, which up- to a hormone-refractory stage [6]. Furthermore, a study sug- regulated p53 protein levels (Figure 4B), resulted in an as- gested that the expression of gain-of-function mutants of p53 sociation of p53 protein with the regulatory region (compare in LNCaP cells results in androgen-independent cell growth lane 7 with 6) of the AR gene. Together, these observations [20]. Because mutations in the p53 gene are associated with indicated that the activation of p53 in LNCaP cells with the progression of PC to advance stages [18–20], our obser- etoposide, which resulted in downregulation of AR expres- vations provide support for the idea that mutations in p53 gene sion, was associated with the binding of p53 protein to the in PC cells contribute to increased expression of AR and the 5V-regulatory region of the AR gene. Together, these obser- progression to androgen independence. vations provide support to the idea that p53 negatively regu- It has been noted that male p53-null mice have less apop- lates the transcription of AR gene in LNCaP cells. tosis 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 re- Figure 6. p53 associates with the 5V-regulatory region of the AR gene. (A) A sulted in downregulation of p21CIP1, and upregulation of Bcl2 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 and AR are consistent with the above observations. Moreover, p53/ from the p53 DNA – binding consensus sequence. (B) The 5V-regulatory re- we noted that levels of AR were detectable in HCT116 gion of human AR gene indicating the locations of the potential p53 DNA – cells, but not in isogenic HCT116p53+/+ cells. Together, these binding site (nucleotides 5290 – 5309), two primers that were used for PCR (after chromatin precipitation), and the mRNA start site (nucleotide 5778) in observations support the idea that p53 negatively regulates the AR gene (using the reference GenBank accession no. X78592). (C) the expression of AR in LNCaP and HCT116 cells. Chromatin was prepared from LNCaP cells either left untreated or treated Previous studies have revealed that increased levels of 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 endogenous as well as exogenous p53 in PC cells lead to antibody (lanes 4 and 5). DNA was extracted from immunoprecipitates and growth inhibition and apoptosis in vitro and in vivo [19, PCR-amplified using a pair of primers that flanked the p53 DNA – binding site 47–49]. Because expression of AR is important for the 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 proliferation and survival of PrECs [39,40] and increased cells. As a negative control, we did not add any DNA in the PCR (lane 1). expression of AR is associated with androgen independence

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