Androgen Induction of Cyclin-Dependent Kinase Inhibitor p21 Gene: Role of Androgen Receptor and Transcription Factor Sp1 Complex

Shan Lu, Guido Jenster and Daniel E. Epner

Department of Medicine (S.L., D.E.E.) Baylor College of Medicine Houston, Texas 77030
Department of Urology (G.J.) Erasmus University Rotterdam, The Netherlands


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous studies have shown that androgen up-regulates expression of the p21 (WAF1, CIP1, SDI1, CAP20) gene, which contains a canonical androgen response element (ARE) in its proximal promoter region. We undertook the current studies to determine whether elements in the p21 promoter other than the ARE mediate androgen action. We found that deletion of the ARE did not completely abolish the promoter responsiveness to androgen, suggesting that additional cis-regulatory elements within the p21 core promoter may also be involved in androgen responsiveness. The p21 core promoter is GC-rich and contains six binding sites for transcription factor Sp1. We determined whether one or more of these Sp1 sites mediate androgen responsiveness of the p21 promoter. To do so, we used a transient transfection assay with p21 promoter-luciferase reporter constructs. The reporter activity of a construct lacking the ARE but containing all six Sp1 sites was induced approximately 3-fold by androgen. Mutation of Sp1–3 nearly eliminated basal promoter activity as well as androgen responsiveness, whereas deletion of Sp1–1 and Sp1–2 sites and mutation of Sp1–4, Sp1–5, and Sp1–6 sites had relatively little effect. We also used the mammalian one-hybrid assay and coimmunoprecipitation assay to show that androgen receptor (AR) and transcription factor Sp1 interact with one another. The current studies suggest a model in which AR and transcription factor Sp1 not only bind to their respective consensus sites within the p21 promoter, but also complex with one another, thereby recruiting coactivators and general transcription factors and inducing p21 transcription.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Androgen is essential for prostate development and homeostasis (1, 2). Androgen withdrawal by castration causes massive apoptosis in prostatic epithelial cells within a few days (3, 4). Androgen, acting through androgen receptor (AR), regulates a series of androgen target genes (5, 6). The best characterized androgen-responsive genes are prostate-specific antigen (PSA) and kallikrein-2 (KLK-2) (7), C(3) protein (8), sex-limited protein (Slp) (9), and probasin (10). Androgen response elements (ARE) have been identified in the promoters of these genes. However, none of these genes are known to be involved in maintaining viability and integrity of the prostate epithelial cells. The molecular mechanisms by which androgen maintains viability of the prostate epithelial cells have therefore not been fully elucidated.

Recent studies indicate that androgen regulates several cell cycle-regulatory molecules within prostate epithelial cells. For instance, androgen increases expression and enzymatic activities of cyclin-dependent kinases (CDK) 2 and 4, molecules that generally promote cell proliferation (11). Androgen has variable effects on CDK inhibitors: it inhibits expression of p16 (MTS1, CDKN2), a member of the p16 family of CDK inhibitors (11), whereas it increases expression of p21 (WAF1, CIP1, SDI1, CAP20), which belongs to a second family of CDK inhibitors (12). p21 was first identified as a CDK inhibitor and later was found to be involved in various biological processes, such as cell cycle control, DNA repair, and antiapoptosis (13, 14, 15). p21 induces cell cycle arrest in response to DNA damage and protects cancer cells against p53-mediated apoptosis. These studies revealed a novel class of genes involved in an androgen-signaling pathway.

Regulation of p21 expression has been studied extensively. p21 gene is induced by p53 (16), transforming growth factor-ß (TGF-ß) (17), signal transducer and activator of transcription 1 (STAT1) (18), vitamin D (19), and nerve growth factor (NGF) (20). The corresponding cis-regulatory elements for each of these factors have been identified in the p21 promoter. We recently found that p21 expression is also activated by androgen via a canonical ARE in its proximal promoter region (12). Androgen up-regulates expression of the p21 gene in stimulating prostate cancer cell proliferation, indicating that the major function of p21 in the prostatic epithelial cells is not cell cycle block. In situ hybridization studies showed that all prostatic epithelial cells, but only a few stroma cells, express p21 protein (21). The functional significance of p21 expression in the prostatic epithelial cells in an androgen-regulated manner is not clear.

The present studies were carried out to further investigate the molecular mechanisms of androgen induction of the p21 gene. We found that, in addition to a canonical ARE in the core promoter of the p21 gene, Sp1 binding sites are also involved in the induction of p21 by androgen. We also present evidence that transcription factor Sp1 and androgen receptor (AR) interact with one another. These findings suggest a novel molecular mechanism by which androgen regulates expression of its target genes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sp1 Sites Are Involved in Androgen Responsiveness of the p21 Gene
In previous studies we identified a canonical ARE at -200 bp position of the p21 gene promoter (12). Using electrophoresis mobility shift assay, we found that AR bound directly to the ARE. Wild-type ARE, but not mutated ARE, conferred androgen responsiveness to a heterologous promoter, suggesting that the ARE is functional.

In the current study, we determined whether cis-regulatory elements in the p21 core promoter in addition to the ARE mediate androgen action. To do so, we used transient transfection assays with various p21 promoter-luciferase reporter constructs. We found that construct p21(-215)-Luc containing 215 bp of the p21 promoter, including the ARE, conferred 6.2-fold induction by androgen in AR-positive LNCaP-FGC cells (Fig. 1Go). Elimination of the ARE did not completely eliminate androgen responsiveness. Construct p21(-190)-Luc, which contained 190 bp of the p21 promoter but lacked the ARE, retained 2.8-fold induction by androgen. We therefore hypothesized that the p21 promoter contains cis-regulatory elements in addition to the ARE that mediate androgen responsiveness.



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Figure 1. Determination of ARE-Dependent and -Independent Induction of the p21 Promoter Activity by Androgen

A, Core promoter sequence of the p21 gene. ARE is boxed. Six Sp1 binding sites are underlined. TATA box is in bold. B, The upper panel illustrates the two reporter constructs driven by the p21 promoter. Results of transient transfection assay are shown in the lower panel. LNCaP-FGC cells (105 cells per well in 12-well plate) were transiently transfected with either luciferase reporter construct p21(-215)-Luc (0.3 µg/well plasmid DNA) or construct p21(-190)-Luc (0.3 µg/well plasmid DNA). Subsequently, cells were treated with 10-8 M of AR agonist R1881 for 48 h in RPMI-1640 medium supplemented with 10% stripped FBS, followed by luciferase assay. Each sample was normalized by protein concentration. C, Core promoter sequence of the human tissue transglutaminase gene. D, The upper panel illustrates the two reporter constructs. The lower panel shows the transient transfection results of luciferase reporter constructs pXP2-TG-Luc, pXP2-{Delta}CAAT-Luc, and bRE2-TK-Luc (0.3 µg/well plasmid DNA).

 
Sequence analysis of the p21 core promoter revealed that the 190 bp promoter fragment is GC rich and includes a TATA box and six Sp1 sites (Fig. 1AGo). We next investigated whether the Sp1 sites play a role in androgen induction of the p21 gene. To do so, we used a series of reporter constructs containing the p21 promoter sequence spanning -93 to +1 bp, in which 10-bp nucleotide blocks were progressively mutated (Fig. 2Go). We found that construct p21P93-s conferred approximately 4-fold induction by androgen, indicating that the cis-elements involved in androgen responsiveness are included in -93 ~ +1 bp sequence and that Sp1–1 and Sp1–2 sites are not critical for this effect. Construct p21P93-s mut2, in which the Sp1–3 binding site was destroyed, showed a dramatic decrease of basal promoter activity and minimal androgen responsiveness. In contrast, disruption of Sp1–4, Sp1–5, and Sp1–6 sites had relatively little effect on androgen responsiveness or basal activity (Fig. 2Go), suggesting that the Sp1–3 site is the major cis-element other than the ARE contributing to the basal promoter activity and androgen responsiveness of the p21 gene. Mutation of the TATA box did not eliminate the basal promoter activity and androgen responsiveness of the p21 promoter, suggesting that the TATA box is not critical for the promoter activity.



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Figure 2. Mutational Analysis of Sp1 Binding Site Involved in Induction of the p21 Gene by Androgen

The left panel illustrates luciferase reporter construct p21P93-s-Luc containing 93 bp of the p21 core promoter sequence, as well as mutants with progressive mutation of a 10-bp block spanning from -93 bp to -34 bp of the core promoter sequence. The right panel shows results of corresponding transient transfection assays. LNCaP-FGC cells were transiently transfected with luciferase reporter constructs (0.3 µg/well plasmid DNA) p21P93-s, p21P93-s mut1, p21P93-s mut2, p21P93-s mut3, p21P93-s mut4, p21P93-s mut5, or p21P93-s mut6, respectively. The cells were treated with AR agonist R1881 (10-8 M) for 48 h, followed by luciferase assay.

 
We next determined whether involvement of Sp1 sites in androgen induction is specific for p21 gene. The human tissue transglutaminase gene, like p21, has GC-rich promoter including a TATA box and was used for this purpose (Fig. 1CGo) (22). As shown in Fig. 1DGo, both reporter constructs pXP2-TG-Luc and pXP2-{Delta}CAAT-Luc, each containing four Sp1 sites with or without a CAAT box, did not respond to androgen stimulation. For an additional control, minimal TK promoter construct (bRE2-TK-Luc) linked with two tandem repeats of retinoid acid response element (RARE) also did not respond to androgen treatment. These data suggested that enhanced GC-rich core promoter activity conferred by androgen stimulation is specific for the p21 promoter.

Interaction between Transcription Factor Sp1 and AR in Androgen Induction of the p21 Gene Determined by Mammalian One-Hybrid Assay
Since both ARE and Sp1 binding sites in the p21 proximal promoter were found to be involved in induction of the gene by androgen, we then used the mammalian one-hybrid assay to investigate whether this effect is mediated by interaction between AR and transcription factor Sp1 in a ligand-dependent manner. The mammalian one-hybrid system has two components: 1) a transcription factor that binds DNA and functions as bait, and 2) a coactivator that interacts with the bait and also serves a bridging function. The bridging function of the coactivator results in recruitment of other coactivators and general transcription factors to form a preinitiation complex, resulting in transcription of the reporter gene. In our system, transcription factor Sp1 functioned as bait and bound DNA at the Sp1 site(s). Coactivators consisted of fusion proteins between full-length AR or the ligand binding domain (ARLBD) of AR with the transactivation domain from either viral protein 16 (VP16) or p65/RelA (p65) (Fig. 3AGo) (23). This system resulted in androgen-dependent transactivation by these fusion proteins.



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Figure 3. Interaction between Transcription Factor Sp1 and AR as Determined by Mammalian One-Hybrid Assay

A, Illustration of the fusion protein constructs. Constructs pcDNA-ARo-VP16 and pcDNA-ARo-p65 contained full-length AR cDNA fused to the transactivation domain of either VP16 or p65/RelA protein at the C terminus of the AR gene. Constructs pcDNA-ARLBD-VP16 and pcDNA-ARLBD-p65 included the ligand-binding domain of AR fused to the transactivation domain of either VP16 or p65/RelA protein at the C terminus of the ligand-binding domain. B, Mammalian one-hybrid assay. LNCaP-FGC cells were transiently cotransfected with 0.3 µg/well of p21P93-s reporter and 0.3 µg/well of either control plasmids CMV vector, pcDNA-VP16, pcDNA-p65, pcDNA-ARLBD, or fusion proteins pcDNA-ARo-VP16, pcDNA-ARLBD-VP16, pcDNA-ARo-p65, pcDNA-ARLBD-p65, respectively. The cells were treated with R1881 (10-8 M) for 48 h, followed by luciferase assay.

 
LNCaP-FGC cells were used for the mammalian one-hybrid assay, since they express high levels of transcription factor Sp1 (data not shown). The cells were transiently cotransfected with p21P93-s reporter construct and fusion proteins, respectively. As demonstrated in Fig. 3BGo, fusion proteins pcDNA-ARo-VP16 and pcDNA-ARo-p65 containing full-length AR sequence conferred 22.2-fold and 17.0-fold induction of the p21 promoter activity by androgen, respectively. In contrast, the control vectors pcDNA-VP16, pcDNA-p65, and pcDNA-ARLBD containing either VP16 domain, p65 domain, or ARLBD domain, respectively, as well as CMV empty vector, showed only 5- to 7-fold induction by androgen. This relatively low level induction was probably due to endogenous transcription factor Sp1 and AR interaction. These results demonstrated that the fusion proteins containing full length AR enhanced p21 promoter activity in a ligand-dependent manner, suggesting that transcription factor Sp1 and AR proteins complex with one another.

We then investigated whether the ligand-binding domain of AR alone can interact with transcription factor Sp1. The mammalian one-hybrid assay showed that fusion proteins pcDNA-ARLBD-VP16 and pcDNA-ARLBD-p65 containing only the ligand-binding domain conferred 12.6- and 98.3-fold induction by androgen, respectively (Fig. 3BGo). These data indicated that AR binds to transcription factor Sp1 via the ligand-binding domain of AR. However, domains other than the ligand-binding domain of AR may also be involved in binding transcription factor Sp1.

We next measured the affinity of two of the fusion proteins for AR agonist R1881. R1881 at a concentration of 10-10 M dramatically increased the reporter activity driven by pcDNA-ARo-VP16 containing full length of AR cDNA (Fig. 4AGo). The EC50 of this fusion protein was approximately 5 x10-10 M of R1881, which is similar to that of endogenous AR in LNCaP-FGC cells as measured by transactivation of the p21 promoter in a transient transfection assay (12). In contrast, the fusion protein pcDNA-ARLBD-p65, consisting of the AR ligand-binding domain fused to the transactivation domain of p65, had a low affinity for androgen with a EC50 of 10-9 M (Fig. 4BGo). Despite its low affinity for androgen relative to pcDNA-ARo-VP16, pcDNA-ARLBD-p65 had a greater transactivation activity in the mammalian one-hybrid assays (Fig. 3BGo).



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Figure 4. Dose-Response Curve of the Fusion Proteins

LNCaP-FGC cells were transiently cotransfected with 0.3 µg/well of reporter p21P93-s and 0.3 µg/well of either fusion protein pcDNA-ARo-VP16 (A) or pcDNA-ARLBD-p65 (B). The various concentrations of R1881 were added to the samples for 48 h, followed by luciferase assay.

 
We again used the mammalian one-hybrid system to determine which region of the p21 promoter other than the ARE is activated by androgen induction. The fusion protein pcDNA-ARLBD-p65 (Fig. 3AGo) was used for these experiments, since it was found to have the greatest transactivation function for wild-type p21 promoter as compared with other fusion proteins (Fig. 3Go). The reporter construct p21P93-s mut2, which contains a mutation within Sp1–3 site, showed a low basal promoter activity and a minimal response to ligand stimulation (Fig. 5Go). In contrast, the wild-type construct p21P93-s, as well as constructs containing mutations within other Sp1 sites, maintained high level induction by androgen (Fig. 5Go). These results confirmed the critical role of the Sp1–3 site in maintaining basal activity and androgen inducibility of the p21 promoter, as suggested by previous experiments (Fig. 2Go).



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Figure 5. Determination of Sp1 Sites Involved in Androgen-Mediated Induction of the p21 Gene by Mammalian One-Hybrid Assay

LNCaP-FGC cells were transiently transfected with 0.3 µg/well of fusion protein pcDNA-ARLBD-p65 and 0.3 µg/well of either p21 promoter reporter construct p21P93-s or its mutants p21P93-s mut1, p21P93-s mut2, p21P93-s mut3, p21P93-s mut4, p21P93-s mut5, or p21P93-s mut6, respectively. The cells were treated with R1881 (10-8 M ) for 48 h, followed by luciferase assay.

 
Interaction between Transcription Factor Sp1 and AR Determined by Coimmunoprecipitation
Although the mammalian one-hybrid assay strongly suggested that AR and transcription factor Sp1 complex with one another, we used coimmunoprecipitation followed by Western blot analysis to further test this possibility. Nuclear extracts were prepared from control and androgen-treated LNCaP-FGC cells and used for coimmunoprecipitation. As expected, AR translocated from cytosol to nucleus upon androgen stimulation was demonstrated by an increased immunoprecipitated AR protein level in the nuclear extract (Fig. 6AGo). AR also coimmunoprecipitated with transcription factor Sp1 using anti-Sp1 antibody for immunoprecipitation, as indicated by a detected band corresponding to AR protein in response to androgen stimulation (Fig. 6AGo, lane2). Reciprocally, transcription factor Sp1 was coimmunoprecipitated with AR using anti-AR antibody for immunoprecipitation, indicated by an enhanced precipitated Sp1 protein upon androgen stimulation (Fig. 6BGo). Nuclear alteration of transcription factor Sp1 was not observed by androgen stimulation. These data convincingly demonstrated the interaction of AR and transcription factor Sp1.



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Figure 6. Determination of Interaction between AR and Transcription Factor Sp1 by Coimmunoprecipitation Assay

Nuclear extracts from control (lanes 1 and 3) and androgen-treated (lanes 2 and 4) LNCaP-FGC cells were used for coimmunoprecipitation with either anti-Sp1 antibody (lanes 1 and 2) or anti-AR antibody (lanes 3 and 4). Subsequently, the samples were fractionated by SDS-PAGE, followed by Western blot analysis using either anti-AR antibody (A) or anti-Sp1 antibody (B).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the current studies, we demonstrated that induction of the p21 gene by androgen is mediated not only by an ARE within the p21 proximal promoter, but also by one or more Sp1 binding sites. Of the six Sp1 binding sites in the core p21 promoter region, the Sp1–3 site was most critical for maintaining basal promoter activity and androgen responsiveness. Our studies are consistent with previous ones demonstrating that Sp1 sites in the p21 core promoter are also important for induction by several other factors via different signaling pathways. The Sp1–1 and Sp1–2 sites were involved in 12-O-tetradecanoylphorbol-13-acetate (TPA)- and familial breast cancer susceptibility (BRCA-1)-mediated p21 induction (24, 25), while the Sp1–3 site was required for p21 induction by progesterone receptor (26) and TGF-ß (17).

Using the mammalian one-hybrid system, we also found that AR and transcription factor Sp1 interact with one another. Based on these results, we hypothesize that AR and transcription factor Sp1 form a complex with one another upon binding to their respective sites within the p21 promoter. Formation of this complex in response to androgen could facilitate binding of other coactivators and general transcription factors to form a preinitiation complex for gene transcription (Fig. 7Go). The consequence of this effect would be to enhance expression of the androgen target gene p21.



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Figure 7. The Proposed Composite Effect of ARE and Sp1 Site in Induction of the p21 Gene by Androgen

Conformational change of AR upon androgen binding enables the receptor to bind to ARE and interact with transcription factor Sp1. This functional interaction between AR and transcription factor Sp1 facilitates assembly of transcriptional coactivators and general transcription factors (GFTs) into a transcription preinitiation complex, resulting in an enhanced expression of the p21 gene. The ARE, TATA box, and six Sp1 binding sites in the core promoter region of the p21 gene are illustrated.

 
Our results suggest an alternative mechanism of androgen action on target gene expression. Ligand-bound steroid hormone receptors are classically thought to regulate gene expression by binding to the consensus hormone response elements in target genes (27). However, several recent studies have shown that some hormone-responsive genes do not contain the corresponding hormone response elements within their promoters and can be induced via Sp1 sites by steroid hormone receptor and transcription factor Sp1 interaction. For instance, estrogen induces expression of cathepsin D (28), heat shock protein 27 (29), retinoic acid receptor {alpha}1 (30), vitellogenin A1 (31), and rabbit uteroglobin (32) genes, despite the fact that none of the corresponding promoters contain consensus estrogen response elements. Progesterone similarly induces expression of the p21 gene, which does not contain a consensus progesterone response element within its promoter (26). In the current studies, we found that deletion of the ARE in the p21 promoter did not completely eliminate androgen responsiveness, whereas mutation of the Sp1–3 site did. Our studies and previous ones therefore suggest that steroid hormone receptors, such as ER, PR, and AR, are capable of activating transcription of target genes by forming a complex with transcription factor Sp1 and binding Sp1 sites in a ligand-dependent manner rather than by binding classical hormone response elements.

Cross-talk between pathway-specific transcription factors has been demonstrated in various systems (33, 34). Nuclear hormone receptors are engaged in cross-talk with many other transcription factors, such as AP1, nuclear factor-{kappa}B/Rel, Stat, and C/EBP. Recently, with cloning and characterization of transcription coactivators and corepressors, the molecular mechanisms of transcriptional cross-talk between transcription factors became even more complicated. A few factors are needed to be considered for synergism or negative interference of transcriptional cross-talk, including direct interaction between transcription factors, indirect interaction through "bridging protein," and competition for limiting amounts of cofactors, promoter context, and genetic background of cells. For example, interaction of GR and transcription factor AP1 is involved in regulation of the mouse proliferin gene through a "composite glucocorticoid response element" in a cell type-specific fashion (35). When AP1 is composed of c-Fos and c-Jun heterodimer, negative interference by GR will be observed; and when AP1 is a homodimer of c-Jun, synergism is detected. Protein-protein interaction plays a major role in transcriptional cross-talk (33, 34). Mutational analysis has mapped the domains involved in cross-talk to the DNA-binding domain of the nuclear receptor and the bZip domain of AP1. Positive regulations between transcription factors are demonstrated in genes, such as ovalbumin gene by ER, and Fos-Jun complex (36) and rat tryptophan gene by GR and CACCC-box binding factors (37). Our present study showed a positive interaction between AR and transcription factor Sp1 on regulation of the p21 gene. The detailed molecular mechanisms of this regulation remain to be determined.

Androgen impacts almost every organ in the body and can induce expression of many genes (5, 6). However, AREs were identified in only a few gene promoters. It is possible that androgen induces some of its target genes only via the Sp1 site when ARE is absent in these target gene promoters. This possibility will need to be confirmed with future studies.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
Human metastatic prostate adenocarcinoma cell line LNCaP-FGC (American Type Culture Collection, Manassas, VA) was maintained in RPMI-1640 (Life Technologies, Inc., Gaithersburg, MD) supplemented with either 10% FBS or 10% charcoal/dextran treated (stripped) FBS (HyClone Laboratories, Inc. Logan, UT) at 37 C in 5% CO2.

Reagents
R1881 was purchased from DuPont Merck Pharmaceutical Co. (Wilmington, DE).

Plasmids
p21-(-215)-Luc luciferase reporter vector includes a core promoter sequence of the p21 gene spanning from -215 to +1 bp (12). Construct p21(-190)-Luc was generated by inserting a PCR fragment spanning -190 to +1 bp of the p21 promoter to a luciferase reporter vector. Construct p21P93-S containing 93 bp p21 core promoter sequence and its mutants p21P93-s mut1, p21P93-s mut2, p21P93-s mut3, p21P93-s mut4, p21P93-s mut5, and p21P93-s mut6 were gifts from Dr. Xiao-Fan Wang (Department of Pharmacology, Duke University Medical Center, Durham, NC). Constructs pcDNA-ARo-VP16, pcDNA-ARo-p65, pcDNA-ARLBD-VP16, pcDNA-ARLBD-p65, pcDNA-VP16, pcDNA-p65, and pcDNA-ARLBD have been described previously (23).

Transient Transfection Assay
LNCaP-FGC cells (105) were seeded in 12-well tissue culture plates. Next day, lipofectin-mediated transfection was used for the transient transfection assay according to the protocol provided by Life Technologies, Inc. Cell extracts were prepared according to in vitro luciferase assay kit (Promega Corp., Madison, WI). Luciferase assays were performed in a Monolight 2010 Luminometer (Analytical Luminescence Laboratory, San Diego, CA). For each assay, cell extract (20 µl) was added into a cuvette, and the reaction was started by injection of 100 µl luciferase substrate. Each reaction was measured for 10 sec in the Luminometer. Luciferase activity was defined as light units per mg protein.

Mammalian One-Hybrid Assay
Mammalian one-hybrid assay was performed to determine transcription factor Sp1 and AR interaction. Transcription factor Sp1 was used as bait. Artificial coactivators are fusion proteins that contain either full-length AR or the ligand-binding domain of AR fused with transactivation domain from either viral protein 16 (VP16) or mammalian transcription factor p65/RelA.

Coimmunoprecipitation Assay
Nuclear extracts were prepared from control and androgen-treated LNCaP-FGC cells. Coimmunoprecipitation was performed in a modified RIPA buffer (50 mM Tris·Cl (pH 7.5), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml of pepstatin, leupeptin, and aprotinin each). The antibody and protein complexes were precipitated by Protein G plus/protein A agarose beads (Calbiochem, San Diego, CA) at 2500 rpm for 5 min. The beads were washed with the modified RIPA buffer four times. The protein complexes were subjected to Western blot analysis using anti-Sp1 or anti-AR antibody, respectively.


    ACKNOWLEDGMENTS
 
We thank Dr. Zafar Nawaz (Department of Cell Biology, Baylor College of Medicine) for great discussion about this project.


    FOOTNOTES
 
Address requests for reprints to: Dr. Daniel E. Epner, Veterans Affairs Medical Center, Medical Service (111H), 2002 Holcombe Boulevard, Houston, Texas 77030,

This work was supported by NIH Postdoctoral Fellowship 1 F32 CA-80333–01, NIH Grant R29 CA-78355, and the Department of Veterans Affairs.

Received for publication October 22, 1999. Revision received January 18, 2000. Accepted for publication February 7, 2000.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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