Androgen Regulation of the Cyclin-Dependent Kinase Inhibitor p21 Gene through an Androgen Response Element in the Proximal Promoter

Shan Lu, Min Liu, Daniel E. Epner, Sophia Y. Tsai and Ming-Jer Tsai

Department of Cell Biology (S.L., M.L., S.Y.T., M.-J.T.) and Department of Medicine (D.E.E., M.-J.T.) Baylor College of Medicine Houston, Texas 77030


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Androgen is essential for the physiological maintenance of the integrity of prostatic epithelial cells, and castration causes the cells to undergo apoptosis. To study the molecular mechanism of androgen-dependent cell growth, we showed that androgen up-regulates the expression of the cyclin-dependent kinase inhibitor p21 (WAF1, CIP1, SDI1, CAP20) gene at both the mRNA and protein levels. Nuclear run-on assays demonstrated that androgen stimulates endogenous p21 gene expression at the transcriptional level. Transient transfection experiments showed that androgen can enhance the activity of a 2.4-kb promoter of the p21 gene linked to a luciferase reporter. These results suggested that a putative androgen response element (ARE), which mediates androgen response to enhance the p21 transcription, is included in the 2.4-kb promoter fragment. Deletion analysis of the promoter revealed a functional ARE (AGCACGCGAGGTTCC) located at -200 bp of the p21 gene proximal to the promoter region. Electrophoretic mobility shift assay further demonstrated that the androgen receptor specifically binds to this element. Wild-type ARE, but not mutant ARE, confers androgen responsiveness to a heterologous promoter. The up-regulation of p21 gene expression by androgen suggests that p21 may have an antiapoptotic function in prostatic epithelial cells. However, this hypothesis will need to be tested in future experiments.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of the cell cycle is essential for a cell to determine whether it will undergo proliferation, differentiation, or cell death (1). The cell cycle is controlled by the sequential activation of cyclin-dependent kinases (CDKs) upon association with their partner cyclins and the subsequent phosphorylation and dephosphorylation of the CDKs. Checkpoint regulation of the cell cycle is effected by CDK inhibitors (CKIs), of which two classes have recently been identified. The first class of CDK inhibitors comprises p16 (also called MTS1, INK4a) (2), p15 (also called MTS2, INK4b) (3), p18 (4), and p19 (5). Each of these genes encodes a protein with ankyrin-like repeats that specifically inhibits CDK4 and CDK6. Mutations and deletions of p16 and p15 genes have been found to be associated with tumors, suggesting a tumor suppressor function for this class of genes (6, 7, 8, 9). The second class of CDK inhibitors consists of p21 (also called WAF1, CIP1, SDI1, CAP20) (10, 11, 12), p27 (also called KIP1) (13, 14), and p57 (also called KIP2) (15, 16), which possess considerable sequence similarity and can inhibit all known CDK subtypes. This class of genes is mainly involved in development and differentiation (17, 18).

Androgens, acting through their receptors (ARs), are essential for the maintenance of prostatic epithelial cell proliferation and differentiation during development (19). The rates of epithelial cell growth and death in adult prostate glands are in equilibrium, such that there is no net change in cell growth. In an adult rat, the glandular epithelial cells constitute approximately 80% of the total cells in the ventral prostate, and approximately 70% of these cells die by 7 days postcastration (20, 21). The molecular mechanisms of the androgen-mediated maintenance of the integrity of prostatic epithelial cells have not been elucidated and remains an area of active research.

There are many androgen-regulated genes (22). However, only a few of the genes, such as PSA and KLK-2 (23), C(3) protein (24), Slp (25), and probasin (26), have been well characterized, and androgen response elements (AREs) in these genes were identified. Whether these characterized genes are involved in mitogenic signaling of androgen is still unknown. In studying the molecular mechanisms of androgen action on cell growth, we were the first to demonstrate that androgen stimulates the expression of cell cycle genes CDK2 and CDK4 and represses the expression of CDK inhibitor p16 gene, resulting in increased CDK kinase activities (27). Those studies revealed a new class of androgen target genes. In addition, those observations suggested a possible signaling pathway by which androgen stimulates prostate cell growth. In the current study, we identified the CDK inhibitor, p21, as a target gene of androgen. Androgen induces the expression of the p21 gene in prostate cancer cells through an ARE in the proximal p21 promoter.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Androgen Up-Regulates the Expression of the CDK Inhibitor p21 in LNCaP-FGC Cells
To investigate the molecular mechanisms of androgen-dependent growth of prostatic epithelial cells, we used an androgen-dependent prostatic carcinoma cell line, LNCaP-FGC, as an in vitro model (27). We observed that androgen can up-regulate the expression of the CDK inhibitor p21. After culturing the cells for a week in medium containing 10% stripped FBS, the expression levels of the p21 gene were examined in response to androgen stimulation. As shown in Fig. 1AGo, the level of p21 mRNA in LNCaP-FGC cells was increased after 2 h of treatment with the AR agonist R1881 (10-8 M) and continued to increase thereafter up to 48 h. In contrast, no detectable p21 mRNA was observed in androgen-independent PC-3 cells. The enhancement of p21 expression is specific, since the expressions of CDK inhibitor p27 (KIP 1) is not altered in response to androgen stimulation in both LNCaP-FGC and PC-3 cells. We also found that p21 protein levels in LNCaP-FGC cells, as measured by Western blot analysis, increased in response to R1881 within 8 h (Fig. 1BGo).



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Figure 1. Up-Regulation of the p21 Gene by Androgen

A, After LNCaP-FGC and PC-3 cells were cultured in medium containing 10% stripped FBS for 1 week, R1881, at a concentration of 10-8 M, was added to these cells and the cells were incubated for 0, 2, 8, 24, and 48 h. Subsequently, the total RNAs were isolated, and 20 µg of RNA per sample were used in Northern analysis as described in Materials and Methods. The same filter was used for hybridization with p21, p27, and GAPDH cDNA probes, respectively. Fold increases in the p21 mRNA levels quantified by ScanAnalysis program were indicated under the Northern blot. B, LNCaP-FGC cells were cultured in medium containing 10% stripped FBS for 1 week. Subsequently, R1881 (10-8 M) was added for 2, 8, and 24 h. Total cellular proteins were isolated and subjected to Western blot analysis using 50 µg protein per sample. Ponceau S staining was used as protein loading control. Fold increases of p21 protein levels were indicated under each Western blot. Each Northern or Western analysis experiment has been repeated more than three times with sample collected at different times, and consistent results were observed. C, After LNCaP-FGC cells were cultured in medium containing 10% stripped FBS for 1 week, cycloheximide (CHX, 50 uM) was added to the cells for 48 h and R1881 (10-8 M) was added to these cells for 0, 2, 8, 24, and 48 h. Subsequently, the total RNAs were isolated and 20 µg of RNA per sample was used in Northern analysis. D, After LNCaP-FGC cells were cultured in medium containing 10% stripped FBS for 1 week, R1881 was added to the cells overnight, and nuclei were isolated from both control and R1881-treated cells. Subsequently, run-on transcription was performed for 30 min followed by RNA isolation. {zeta}-Probe strips containing 500 ng of either p21 cDNA or GAPDH cDNA on each dot slot were used for hybridization probed by labeled RNA from both control and R1881-treated cells.

 
Androgen Up-Regulates the p21 Gene Expression at the Transcriptional Level
To investigate whether androgen-dependent up-regulation of p21 is dependent on protein synthesis, LNCaP-FGC cells were treated with protein synthesis inhibitor, cycloheximide, for 48 h with R1881 stimulation for various times. Northern blot analysis showed that cycloheximide superinduces the expression of p21 gene with or without R1881 treatment, and the enhanced expression of p21 gene in response to androgen stimulation was not observed in the presence of cycloheximide (Fig. 1CGo). It is possible that this phenomenon is due to an enhanced p21 mRNA stability stimulated by cycloheximide by an unknown mechanism. We next did nuclear run-on assays to determine whether androgen directly stimulates p21 gene transcription. As shown in Fig. 1DGo, we found an enhanced mRNA synthesis in the RNA preparation isolated from R1881-treated nuclei as compared with the RNA isolated from control nuclei. As a control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expressions are similar between control and R1881-treated sample.

To examine whether androgen directly up-regulates the expression of the p21 gene, transient cotransfection experiments were performed. An AR expression vector (CMV-AR) and a reporter construct (p21(-2400)-Luc) containing 2.4 kb of the p21 promoter fused to a luciferase reporter were cotransfected into COS 1 cells. Subsequently, the reporter luciferase activity in response to androgen stimulation was determined. AR agonist R1881 (10-8 M) induces an approximately 2-fold increase in luciferase activity in COS 1 cells cotransfected with the p21(-2400)-Luc and cytomegalovirus (CMV)-AR vectors (Fig. 2AGo). There is no increase in luciferase activity upon androgen stimulation in COS 1 cells transfected with p21(-2400)-Luc vector without the AR expression vector. This result suggests that the COS 1 cells contain no, or low levels of, endogenous AR. As a control, a reporter construct, RARE-tk-Luc, which contains two copies of retinoic acid response element (RARE) fused upstream of the minimal thymidine kinase (tk) promoter, fails to respond to R1881 when cotransfected with AR expression vector in COS 1 cells. Since these experiments were carried out in the COS 1 cell line, which is monkey kidney cells, the results may not reflect the physiological condition. Thus, these experiments were also carried out in prostatic carcinoma LNCaP-FGC cells, which contain a high expression level of a gain-of-function mutated form of endogenous AR. Figure 2Go showed that an increased expression of luciferase activity was also observed in LNCaP-FGC cells transfected with p21(-2400)-Luc vector alone in response to R1881 (Fig. 2BGo). These results indicate that androgen-dependent induction of luciferase reporter gene expression is specifically mediated by AR, and a functional ARE is likely to reside in the 2.4-kb fragment of the p21 promoter.



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Figure 2. Determination of Androgen Responsiveness of the p21 Promoter by Transient Transfection Assay

A, COS 1 cells (105) were seeded in six-well tissue culture plates. The next day, the cells were cotransfected with 0.25 µg of p21(-2400)-Luc or RARE-tk-Luc vector with or without 0.25 µg CMV-AR expression vector. The cells were stimulated with 10-8 M R1881 for 48 h. Cell extracts were prepared according to an in vitro luciferase assay kit followed by luciferase assay as described in Materials and Methods. B, LNCaP-FGC cells (2 x 105) were seeded in six-well tissue culture plates. The next day, the cells were transfected with 0.25 µg p21(-2400)-Luc vector for the transient transfection assay. The error bar represents the average of duplicate samples of each experiment. Each transient transfection assay has been repeated more than five times.

 
Characterization of a Functional ARE in the p21 Promoter
To identify the ARE(s) in the p21 promoter, a series of progressive 5'-promoter deletion mutants were generated (Fig. 3AGo). COS 1 cells were used for transient transfection experiments by cotransfecting these p21 deletion constructs and CMV-AR expression vector. As shown in Fig. 3AGo, the constructs p21(-2400)-Luc, p21(-1800)-Luc, p21(-600)-Luc, and p21(-215)-Luc, which include 2.4 kb, 1.8 kb, 0.6 kb, and 0.215 kb of the p21 promoter fragment, respectively, showed an increased promoter activity upon androgen stimulation. In contrast, the construct p21(-60)-Luc, which contains only 60 bp of the p21 gene promoter, fails to respond to androgen. These results strongly suggested that the p21 promoter spanning from nucleotides -215 to -60 contains a functional ARE. A dose-dependent induction of the expression of luciferase reporter gene driven by 215 bp of the p21 promoter fragment by varying concentrations of R1881 showed that a concentration as low as 10-11 M can stimulate the reporter gene expression (Fig. 3BGo). The dose-response curve reached a plateau at concentrations higher than 10-10 M of R1881, implying that all binding sites have been saturated. A similar dose-response curve was obtained by transfecting only the reporter construct p21(-215)-Luc into LNCaP-FGC cells with R1881 EC50 at approximately 5 x 10-11 M (data not shown).



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Figure 3. Localization of ARE in the p21 Promoter by Deletion Analysis

A, The recombinant luciferase reporter constructs containing deleted p21 promoter fragments were shown on the left panel. Luciferase activities were determined by transient cotransfection assay. COS 1 cells were cotransfected with 0.25 µg reporter construct with 0.25 µg CMV-AR expression vector for 48 h in the presence and absence of R1881 (10-8 M), followed by luciferase assay as described in Materials and Methods. B, Dose-dependent up-regulation of p21 promoter activity by androgen. COS 1 cells were cotransfected with 0.25 µg of p21(-215)-Luc with 0.25 µg of CMV-AR expression vector for 48 h in the presence of increasing concentrations of R1881.

 
Sequence analysis of the p21 promoter spanning the regions between -215 to -60 bp revealed a likely ARE sequence (AGCACGCGAGGTTCC) located at the -200 bp position of the promoter. This putative ARE is homologous to the AREs found in the promoters of the hKLK2, C(3), PSA, and Slp genes (Fig. 4AGo). Therefore, a double-stranded oligonucleotide containing the putative ARE sequence corresponding to the region from nucleotides -215 to -186 of the p21 gene promoter was used in an electrophoretic mobility shift assay (EMSA) to determine whether AR can bind to this sequence. Figure 4BGo showed that the binding of AR to the oligo probe can be supershifted by anti-AR antibody but not by a nonspecific rabbit IgG, suggesting that AR binds specifically to the oligo probe. Unlabeled wild-type oligonucleotide duplexes including AREs from the p21 gene and C(3) gene could compete efficiently for the binding of AR to the ARE in the p21 gene, indicating that binding is specific. To further support that the binding is specific, multiple ARE mutants were generated by changing the conserved nucleotides of the p21 gene ARE as shown in Fig. 4AGo. EMSAs showed that there is no specific binding of AR to mutant-2 ARE and a drastic decrease in AR binding to mutant-3 ARE as compared with the efficient binding of AR to the wild-type ARE (Fig. 5AGo). Similarly, the specific binding of AR to the wild-type ARE can be competed for the unlabeled wild-type ARE oligo probe of the p21 gene but not by the unlabeled mutant-2 ARE (Fig. 5BGo). These results collectively indicate that mutation of more than one conserved nucleotide in the ARE decreases or abolishes the binding by AR.



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Figure 4. Binding of AR to the ARE in p21 Promoter

A, Sequence comparison of the ARE in the p21 gene with the well characterized AREs in hKLK2, C(3 ), PSA, and Slp genes. Mutated AREs for EMSA are indicated as ARE-mut1, ARE-mut2, and ARE-mut3. B, Cell nuclear extract prepared from LNCaP-FGC cells treated with R1881 was analyzed for AR complex formation with the ARE in the p21 gene in a EMSA. The end-labeled oligonucleotide containing the wild-type ARE in the p21 gene was used as probe. Twenty-fold excess of the unlabeled ARE oligonucleotide probe either from the p21 gene or from the C(3 ) gene was used for binding competition.

 


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Figure 5. Mutational Analysis of Specific Binding of AR to the ARE from the p21 gene in EMSA

A, The end-labeled wild-type and mutated ARE oligonucleotides as indicated in Fig. 4AGo were used as probe for EMSA. B, Dose-dependent competition of AR binding to the wild-type ARE by the unlabeled wild-type ARE oligonucleotide and ARE-mut2 oligonucleotide; 20-, 10-, 5-, 2.5-, and 1.2-fold of unlabeled wild-type or mutated oligonucleotides were added in each binding reaction, respectively.

 
To further determine the physiological relevance of the ARE of the p21 gene, we investigated whether this ARE can confer androgen responsiveness to a heterologous promoter. An oligo containing two wild-type or two mutated ARE sequences separated by six nucleotides from the p21 gene was subcloned into the pXP2-{Delta}NF1-Luc vector containing the human tissue transglutaminase gene minimal promoter (-32 ~ +5 nucleotides). The resulting constructs are ARE-TATA-Luc and AREmut-TATA-Luc vectors, respectively (Fig. 6Go) (28). Transient transfection of these reporter vectors into LNCaP-FGC cells in the presence or absence of R1881 demonstrated that only the wild-type ARE, but not the mutated ARE, can confer the androgen responsiveness to the minimal promoter of the human tissue transglutaminase gene (Fig. 6Go). In conclusion, these results further support the notion that the ARE found in the p21 gene is authentic.



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Figure 6. The Wild-Type but Not Mutated Form of the ARE in the p21 Gene Confers Androgen Responsiveness to a Heterologous Promoter

Two copies of the wild-type or mutated AREs spaced by six nucleotides were subcloned upstream of the human tissue transglutaminase minimal promoter fused with luciferase reporter to generate ARE-TATA-Luc and AREmut-TATA-Luc constructs. LNCaP-FGC cells (2 x 105) were seeded in six-well tissue culture plates. Next day, the cells were transfected with ARE-TATA-Luc or AREmut-TATA-Luc (0.5 µg) for 48 h in the presence and absence of R1881. Cell extracts were prepared according to an in vitro luciferase assay kit followed by luciferase assay as described in Materials and Methods. The error bar represents an average of duplicate samples of each experiment. Each transient transfection assay has been repeated more than five times.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Multiple factors have been demonstrated to regulate the expression of the p21 gene. In stimulating myelomonocytic cell differentiation, vitamin D transcriptionally activates the expression of the p21 gene, and a vitamin D response element (VDRE) was identified at the promoter sequence from -788 to -756 bp (29). The multifunctional growth factor transforming growth factor-ß (TGFß) can cause cell growth arrest by the up-regulation of the expression of the p21 gene through a TGFß-response element (RE) located at -84 ~ -74 bp position of the p21 promoter (30). By inhibiting cell growth in response to epidermal growth factor and interferon-{gamma} treatment, the p21 gene can be up-regulated through STAT1 (signal transducers and activators of transcription). Three STAT-inducible elements (SIEs) in the promoter of the p21 gene were identified, which are located at -640, -2540, and -4183 bp (31). A functional p53-RE was also located at approximately 2.4 kb upstream of the p21 gene-coding region (11). It has also been demonstrated that the E2A gene products, E12 and E47, can up-regulate the p21 gene expression through two E boxes in the proximal p21 promoter to suppress cell growth (32). Here we demonstrated that androgen transcriptionally regulates the expression of the p21 gene. Our studies thus reveal a cross-talk of cell cycle control and androgen action at the molecular level.

Determination of the biological functions ascribed to p21 suggests that p21 is a multifunctional protein playing a key role in the cell cycle control, DNA repair, and antiapoptosis (33, 34, 35). When one p21 molecule binds to one cyclin-CDK complex, the resulting complex is active and can phosphorylate Rb to allow cell cycle to progression. When two or more p21 molecules bind per cyclin-CDK complex, kinase activities are inhibited, and cell cycle progression is blocked. p21 is known to inhibit proliferating-cell nuclear antigen in DNA replication but not in DNA repair, leading to the inactivation of chromosomal replication while allowing DNA damage-responsive repair (36). In addition, it has been shown that during terminal differentiation of myocytes, p21 induction is correlated with the acquisition of an apoptotic-resistant phenotype (34). p21 Is also required for the survival of differentiating neuroblastoma cells (37). More direct evidence is that p21 protects against p53-mediated apoptosis of human melanoma cells (35). Furthermore, a transcriptionally incompetent p53 can induce apoptosis, but not growth arrest, whereas induction of p21, which is a major transcriptional target of p53, can induce growth arrest but not apoptosis (38). Therefore, this evidence suggests that p21 plays a major role in cell proliferation, differentiation, and apoptosis.

To investigate the molecular mechanisms in the development of androgen-independent growth of prostate cancer, we have established an androgen-independent prostatic carcinoma cell line LNCaP-AI by culturing the parental androgen-dependent LNCaP-FGC cell line in medium containing stripped serum. It is interesting to find that there is a drastic increase in the basal expression of the p21 gene in this androgen-independent cell line, although the cells can grow very well in the absence of androgen (S. Lu, S. Tsai, and M.-J. Tsai, unpublished observation). Androgen is essential for the maintenance of the integrity of the prostatic epithelium, and androgen withdrawal results in massive apoptosis of the prostatic epithelial cells and prostate evolution. bcl-2 Was demonstrated to be involved in the development of androgen-independent growth (39, 40). In normal prostatic secretory epithelial cells, there are no detectable levels of bcl-2 expression. The correlation between the up-regulation of the p21 gene and acquisition of androgen-independent cell growth led us to speculate that in the normal prostatic epithelial cells, up-regulation of p21 gene by androgen may play an antiapoptotic role in an androgen-dependent manner but not an inhibitory role in cell cycle progression.

p21 Is the fourth androgen-regulated cell cycle gene we have identified. To investigate the molecular mechanisms of androgen induction of p21 expression, we have performed detailed functional analyses on the p21 promoter. By deletion and mutation analyses, we have defined a consensus ARE sequence located at -200 bp of the p21 promoter that is required for androgen-activated transcription. EMSA with the wild-type and mutated ARE sequences of the p21 gene using nuclear extracts from LNCaP-FGC cells revealed one specific band of retarded mobility, which can be supershifted by anti-AR antibody. The presence of this retarded band is correlated functionally with the ability of the ARE to drive androgen-mediated transcription to a heterologous promoter.

Recent studies have demonstrated that a glucocorticoid response element (GRE)- or progesterone response element (PRE)-like element with consensus sequence 5'-GGA/TACAnnnTGTTCT-3' functions as ARE, which may be due to the homologous properties between AR, GR, and PR. However, AREs in Slp and probasin genes exhibit preference for AR (25, 26). According to the characteristics of the identified ARE, some of them are simple ARE, which typically consists of an imperfect palindrome sequence with a 3-bp spacer between the two half-sites. The examples are the AREs in PSA and hKLK2 genes (23, 41). There are also complex AREs in which multiple elements and the binding of multiple proteins to these elements are required for full androgen-induced activity. These are exemplified in AREs in Slp and probasin genes (25, 26). For the ARE in the p21 gene, it appears to be a simple ARE element.

In summary, our studies show convincingly that androgen acts on the p21 gene. This finding points to an interesting possibility that the CDK inhibitor p21 may be involved in the androgen-mediated antiapoptotic function in the prostatic epithelial cell. Further studies are currently underway to investigate this hypothesis.


    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 (ATCC), 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, Logan, UT) at 37 C in 5% CO2. Human prostate adenocarcinoma cell line PC-3 (ATCC) was cultured in DME/F12 medium (Life Technologies, Inc.) supplemented with either 10% FBS or stripped FBS.

Reagents
R1881 was purchased from Dupont Biotechnology Systems (Boston, MA). Human p27 and p21 cDNA probes were kindly provided by Dr. Wade Harper (Department of Biochemistry, Baylor College of Medicine). The vector RARE-tk-Luc was a gift from Dr. Richard Heyman (Ligand, La Jolla, CA). Anti-AR antibody was a gift from Dr. Nancy Weigel (Department of Cell Biology, Baylor College of Medicine).

Northern Blot Analysis
Total cellular RNAs from control and R1881-treated samples were isolated using Ultraspec RNA isolation reagent (Biotecx Laboratories, Inc., Houston, TX). Total RNA (20 µg/sample) was fractionated on a 1% formaldehyde agarose gel and transferred onto a nylon filter (Hybond-N, Amersham Life Science, Arlington Heights, IL). Northern hybridization was performed using Quikhyb hybridization solution according to the manufacture’s recommendations (Stratagene, La Jolla, CA). Quantitation of each band was performed using ScanAnalysis computer program (BIOSOFT, Ferguson, MO).

Western Blot Analysis
Aliquots of samples with same amount of protein, determined using the Bradford assay (Bio-Rad, Hercules, CA), were mixed with loading buffer [final concentrations of 62.5 mM Tris-HCl (pH 6.8), 2.3% SDS, 100 mM dithiothreitol, and 0.005% bromophenol blue], boiled, fractionated in a 15% SDS-PAGE, and transferred onto a 0.45-µm nitrocellulose membrane (Bio-Rad). The filters were blocked with 2% fat-free milk in PBS and probed with anti-CDK2 and anti-CDK4 antibodies (0.05 µg/ml IgG) (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS containing 0.1% Tween 20 (PBST) and 1% fat-free milk. The membranes were then washed once in PBST and incubated with horseradish peroxidase-conjugated F(ab')2 of goat antirabbit secondary antibody (Bio-Rad) in PBST containing 1% fat-free milk. After washing four times in PBST, the membranes were visualized using the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham Life Science). Quantitation of each band was performed using ScanAnalysis computer program (BIOSOFT).

Nuclear Run-on Assay
LNCaP-FGC cells were cultured in medium containing 10% stripped serum for 1 week. Nuclei were isolated from approximately 5 x 107 of either control cells or cells treated overnight with R1881. Cells were lysed twice in NP-40 lysis buffer [10 mM Tris-Cl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% NP-40] by incubating on ice for 5 min each time, and obtained nuclei were frozen at -80 C in 100 µl of glycerol storage buffer [50 mM Tris-Cl (pH 8.3), 40% (vol/vol) glycerol, 5 mM MgCl2, 0.1 mM EDTA]. To perform nuclear run-on transcription, the frozen nuclei were thawed at room temperature. One hundred microliters of 2x reaction buffer with nucleotides [10 mM Tris-Cl (pH 8.0), 5 mM MgCl2, 0.3 M KCl, 1 mM ATP, 1 mM CTP, 1 mM GTP, and 5 mM DTT] plus 10 µl of 10 mCi/ml [{alpha}-32P]UTP were added. The reaction was incubated 30 min at 30 C with shaking. The nuclei were spun 5 min at 500 x g and 4 C. RNAs were isolated using Ultraspec RNA isolation reagent (Biotecx Laboratories, Inc.). For hybridization, 500 ng of denatured p21 or GAPDH cDNA was used for each dot slot of Zeta-probe membrane (Bio-Rad). The dot-slot strips were prehybridized in Quick-hyb hybridization solution (Stratagene) for 3 h at 68 C. Subsequently, 2 x 106 cpm/ml denatured RNA probe were added and hybridization was performed for 24 h at 68 C. The strips were washed twice in 2x saline sodium citrate (SSC) and 0.1% SDS at room temperature for 15 min and once in 0.2x SSC and 0.1% SDS at 50 C for 15 min followed by x-ray exposure.

Transient Transfection Assay
COS 1 cells (105) or 2 x 105 LNCaP-FGC cells were seeded in six- well tissue culture plates. Next day, lipofectin-mediated transfection was used for the transient transfection assays according to the protocol provided by Life Technologies, Inc.. Cell extracts were prepared according to in vitro luciferase assay kit (Promega, 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/mg protein.

EMSA
EMSA was carried out as described in a bandshift assay system (Promega). Nuclear extract isolated from LNCaP-FGC cells treated with R1881 was used. The oligo probe containing p21-ARE is AAGCTTAGTACGTGATGTTCTAAGCTT. The probes containing mutated AREs are shown in Fig. 4AGo.


    ACKNOWLEDGMENTS
 
We thank Miss Naomi Lee for assistance in manuscript preparation.


    FOOTNOTES
 
Address requests for reprints to: Address requests for reprints to: Dr. Ming-Jer Tsai, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: mtsai{at}bcm.tmc.edu

This work was supported by Baylor Specialized Program of Research Excellence in prostate cancer (CA-58204).

Received for publication June 25, 1998. Revision received October 28, 1998. Accepted for publication November 30, 1998.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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