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Molecular Cancer Research 5, 967-980, September 1, 2007. doi: 10.1158/1541-7786.MCR-06-0318
© 2007 American Association for Cancer Research
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Signaling and Regulation

Increased Expression of Corepressors in Aggressive Androgen-Independent Prostate Cancer Cells Results in Loss of 1{alpha},25-Dihydroxyvitamin D3 Responsiveness

Huei-Ju Ting1, Bo-Ying Bao2, Jay E. Reeder1,3, Edward M. Messing1,3 and Yi-Fen Lee1,3

Departments of 1 Urology, 2 Chemical Engineering, and 3 Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York

Requests for reprints: Yi-Fen Lee, Department of Urology, University of Rochester, 601 Elmwood Avenue, Box 626, Rochester, NY 14642. Phone: 585-275-9702; Fax: 585-756-4133. E-mail: YiFen_Lee{at}urmc.rochester.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Vitamin D has antiproliferative activity in prostate cancer; however, resistance to vitamin D–mediated growth inhibition occurs. To investigate the mechanisms of vitamin D resistance, we screened two prostate cancer sublines of CWR22rv1, CWR22R-1, and CWR22R-2, with differential sensitivity to vitamin D. CWR22R-2 showed less response to the antiproliferative effect of vitamin D than CWR22R-1. The vitamin D receptor (VDR)–mediated transcriptional activity was also decreased in CWR22R-2. We further showed that the DNA-binding ability of VDR was decreased and the amount of NCoR in VDR response element was increased in CWR22R-2. Analysis of VDR-associated protein profiles found higher expression of the corepressors, NCoR1 and SMRT, in CWR22R-2 cells. Treatment with the histone deacetylase inhibitor, trichostatin A, increased vitamin D/VDR transcriptional activity and promoted the antiproliferative effect of vitamin D in CWR22R-2 cells. Targeted down-regulation of NCoR1 and SMRT by small interference RNA was able to restore CWR22R-2 response to vitamin D. Together, we showed that increased NCoR1 and SMRT expression in CWR22R-2 cells resulted in reduced VDR-mediated transcriptional activity and attenuated antiproliferative response to vitamin D. Our data suggest that the integrity of the vitamin D/VDR–mediated signaling pathway is crucial in predicting vitamin D responsiveness and thus provide a rational design to improve vitamin D–based treatment efficacy based on molecular profiles of patients. (Mol Cancer Res 2007;5(9):967–80)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Prostate cancer is the most frequently diagnosed noncutaneous cancer in American men. Although androgen ablation therapy has been a successful treatment, prostate cancer eventually progresses to a hormone refractory stage and loses responsiveness to such therapy. Alternative therapeutic methods to fight hormone refractory cancer are necessary for a growing number of patients. Ever since epidemiologic evidence showed that increasing mortality rates from prostate cancer correlated with low serum levels of 1{alpha},25-dihydroxyvitamin D3 (1,25-VD), the active form of vitamin D (1), there have been increasing numbers of studies to support the antiproliferative effects of 1,25-VD on prostate cancer cells both in vivo and in vitro (2-4). However, loss of response to 1,25-VD among numerous established cell lines, especially progressive prostate cancer cells, has triggered a series of studies aimed at understanding the underlying molecular mechanisms of vitamin D refractoriness (5-7). If the mechanisms of vitamin D resistance in progressive prostate cancer cells could be clarified, individualized 1,25-VD treatment regimens could be devised based on tumor characteristics, and these treatments might benefit many prostate cancer patients.

Various responses to 1,25-VD are observed in LNCaP, PC-3, ALVA 31, and DU145 cells. Among them, one common characteristic of the less responsive cell lines is loss of expression of the androgen receptor. Casodex, an androgen antagonist, can suppress the antiproliferative effect of 1,25-VD in LNCaP cells, indicating that androgen receptor is involved in 1,25-VD signaling (6). Our previous study also showed that the effect of 1,25-VD is diminished after knocking down androgen receptor by RNA interference (8). However, one study using MDA PCa 2a and MDA PCa 2b cell lines showed that androgen-dependent and androgen-independent 1,25-VD signaling pathways exist in different cells (7). In previous studies, overexpression of androgen receptor in ALVA 31 and PC-3 cells did not restore 1,25-VD sensitivity, indicating that factors other than androgen receptor are involved in 1,25-VD sensitivity in these cell lines (5, 9). Furthermore, in the LNCaP subline LNCaP-104R1, which developed an androgen-independent phenotype, 1,25-VD exerts an antiproliferative effect similar to that observed in the parental LNCaP cell line (5). Overall, in androgen refractory prostate cancer cell lines, 1,25-VD may not depend solely on androgen receptor signaling to exert its antiproliferative effect.

Because the major genomic function of 1,25-VD is mediated by the vitamin D receptor (VDR), the differential antiproliferation effect of 1,25-VD on prostate cancer cell lines may result from varying amounts or transcriptional activities of VDR among cells. Indeed, overexpression of VDR cDNA in prostate cancer cell lines (JCA-1, PC-3, or DU145) is sufficient to establish the antiproliferative effect of 1,25-VD (10, 11). On the other hand, the stable transfection of antisense VDR cDNA into ALVA 31 cells abolished the response to vitamin D (12). In contrast, ALVA 31 cells exhibited much less growth inhibition in response to 1,25-VD than LNCaP cells, yet the expression of VDR is higher in ALVA 31 (11). Therefore, it is concluded that VDR is not the only factor mediating the growth inhibition signaling of 1,25-VD. However, there are caveats in comparing two cell lines from different sources that have variable genetic backgrounds; moreover, the androgen-independent phenotype of ALVA 31 resulting from numerous factors complicate the mechanisms involved in the antiproliferative effects of 1,25-VD.

To fully uncover the mechanism of vitamin D resistance, thorough investigations of the vitamin D signaling pathway are essential. 1,25-VD is a hydrophobic steroid that can freely diffuse into cells or be transported by cubilin and megalin, transporter proteins that bind to vitamin D–binding protein and mediate import by endocytosis (13). After entering cells, 1,25-VD can be metabolized by 24-hydroxylase (CYP24) to a less active form, 1,24,25-trihydroxyvitamin D3, and excreted. The expression of CYP24 is induced by 1,25-VD, resulting in a feedback mechanism that controls the activity of 1,25-VD (14). The 1,25-VD–bound VDR recognizes specific response elements within target genes and regulates transcription. Like most members of the steroid receptor family, VDR contains a ligand-binding domain, a DNA-binding domain, and an activation function domain. One important partner of VDR is retinoid X receptor (RXR), which forms a heterodimer with ligand-bound VDR to stabilize the VDR-DNA–binding complex. Over the last decade, several coregulators have been identified and shown to modulate the transcriptional activity of VDR by various mechanisms. Such mechanisms include chromatin remodeling, recruitment of basal transcription machinery, and nuclear transportation. Among these coregulators, NCoR1 and SMRT are corepressors that bind to unliganded VDR and repress its transcriptional activity. SRC-1 and P300/CBP are coactivators that contain intrinsic histone acetylase activity to help in chromatin remodeling that facilitates transcription. Moreover, members of the VDR-interacting protein complex, composed of proteins generally present in many transcription factor complexes, work in concert to activate transcription following the recruitment of basal transcription machinery. In a recent study, we discovered that the transcriptional activity of VDR could be modulated by several androgen receptor coregulators (15). Overall, the varied antiproliferative effect of vitamin D might be due to the increased expression of CYP24, decreased expression of VDR or RXR, VDR polymorphisms, elevated expression of VDR corepressors, or other factors involved in vitamin D signaling as suggested by a recent review (16).

To study the mechanisms involved in varied antiproliferative effect of 1,25-VD in prostate cancer cells, we screened the CWR22rv1 sublines and identified two sublines, CWR22R-1 and CWR22R-2, with various responses to 1,25-VD. Because they are derived from the same parental cell line, differences due to genetic background could be eliminated. Characterization of these two cell lines indicated that they are both androgen independent but differ in invasion ability. By dissecting the mechanisms involved in varying responsiveness to 1,25-VD using these two sublines as a model, we identified the molecules that contribute to the developing vitamin D resistance in aggressive prostate cancer cells.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Identification of Differential Antiproliferative Effects of 1,25-VD on CWR22rv1 Sublines
Previous reports have shown differential antiproliferative effects of 1,25-VD in numerous prostate cancer cell lines, including LNCaP, PC-3, DU145, ALVA 31, MDA PCa 2a, and MDA PCa 2b (7, 11). However, the characteristics and genetic features of those cell lines are different. A comparable cell line pair with differential responses to 1,25-VD is needed for mechanistic studies. After comparing the antiproliferative effect of 1,25-VD in cell lines available in our laboratory, we discovered differential effects between two CWR22rv1 sublines, CWR22R-1 and CWR22R-2. As shown in Fig. 1A , 1,25-VD in charcoal-dextran treated fetal bovine serum (CD-FBS)–supplemented medium exerted 15% growth inhibition on CWR22R-1, but only 4% on CWR22R-2, starting from the 4th day of treatment. As shown in Fig. 1B, cell cycle profiles of 1,25-VD–treated cells were analyzed by flow cytometry to show the various responses in these two sublines. In CWR22R-1 cells, G0-G1 accumulation (from 44 ± 0.5% to 54 ± 1.8%) occurred from 3 days after 1,25-VD treatment compared with ethanol-treated controls. However, 1,25-VD does not have significant effects on the cell cycle profile of CWR22R-2 cells at either 3 or 6 days. This suggests that 1,25-VD might regulate some G0-G1 regulatory proteins to trigger G0-G1 arrest in CWR22R-1; however, this regulation is decreased in CWR22R-2 cells. Because 1,25-VD also inhibits growth by inducing apoptosis, which we did not examine here, G0-G1 arrest only represents partial growth inhibition effect of 1,25-VD. The modest G0-G1 arrest effect of 1,25-VD in CWR22R-1 cells at day 6 is possibly due to the cell density reaching confluency that increases the overall G0-G1 population in every treatment group.


Figure 1
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  		FIGURE 1. 1,25-VD–mediated suppression of growth and accumulation of G1 phase are different between CWR22rv1 sublines. A. CWR22R-1 and CWR22R-2 cell lines were seeded at a density (O.D.) of 5 x 104 per well in 24-well plates and cultured in 10% CD-FBS–supplemented RPMI. After 24 h, cells were treated with ethanol (EtOH) or 100 nmol/L 1,25-VD for 6 d. At days 0, 2, 4, and 6, the MTT assay was done to measure the number of viable cells. Each treatment condition and assay was done in triplicate, and the relative growth obtained by comparing with day 0 was calculated. Points, mean; bars, SD. B. Cells were plated in 100-mm dishes and treated with either ethanol or 100 nmol/L 1,25-VD for 3 and 6 d. Cells were fixed in 70% ethanol and stained with propidium iodide. Cell cycle profiles and distributions were then determined by flow cytometric analysis of 10,000 cells.

 
The origin of these two sublines was verified by single nucleotide polymorphisms (SNP) analysis. A comparison of genotype calls using default variables is presented in Table 1 . Homozygous loci are identified as AA or BB, whereas heterozygous loci are identified as AB. In addition, there were 466 loci in CWR22R-1 and 294 loci in CWR22R-2 with insufficient or ambiguous signals ("No call" in Table 1). Although a "no call" may indicate homozygous deletion, these were not clustered in any particular region of the genome and were concordant in the two cell lines at 209 loci. Disregarding the "no call" loci, there was concordance at the 9650 loci. There were three loci in which a heterozygous locus in one cell line was found to be homozygous in the other cell line. SNP_A-1509275 in IL21R, interleukin 21 receptor gene, was heterozygous in CWR22R-1 and homozygous in CWR22R-2. SNP_A-1514387 in HS3ST1, heparan sulfate (glucosamine) 3-O-sulfotransferase gene, was heterozygous in CWR22R-1 and homozygous in CWR22R-2. Finally, SNP_A-1517295 in KRTHA7, keratin hair acidic 7 gene, was heterozygous in CWR22R-2 and homozygous in CWR22R-1. A specific role for these genes in the observed phenotypic difference is not apparent and they may represent miscalls. As such, the rate for this type of miscall is acceptable at only 3 in 10,204.


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  		Table 1. Summary of SNP Array Genotyping Data

 
Because these two cell lines are derived from the same parental cell line, many confounding factors can be eliminated when studying the mechanisms underlying the variable antiproliferative effects of 1,25-VD.

Aggressive Character of CWR22R-2 Subline Is Shown in Invasion Assay
Further examination and evaluation of the characteristics of the two chosen CWR22rv1 sublines showed that, first, CWR22R-1 cells were larger, formed looser intercellular junctions, and spread out better than CWR22R-2 cells (Fig. 2A ). Second, the cell doubling time was shorter for CWR22R-2 (1.2 days) compared with CWR22R-1 (1.6 days; Fig. 2B). Finally, cell invasiveness was measured to show the aggressiveness of these cell lines. As shown in Fig. 2C, CWR22R-2 was more active in invasion of Matrigel coating. Taken together, we showed that CWR22R-2 cells grew faster and showed enhanced invasiveness; hence, CWR22R-2 had potentially more malignant characteristics than CWR22R-1.


Figure 2
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  		FIGURE 2. General characterization of CWR22rv1 sublines. A. CWR22R-1 and CWR22R-2 cell lines were seeded at a density of 106 cells in 100-mm dishes and cultured in 10% FBS supplemented RPMI. After 2 d, pictures of well-attached cells were taken under x100 magnification. B. Cells were seeded in 24-well plates and cultured in 10% FBS supplemented RPMI. On days 1, 2, and 3, the MTT assay was done. Absorbance (O.D.) values were plotted, and the doubling time of each cell line was calculated. C. Cells were seeded at a density of 105 per chamber in Matrigel-coated inserts and serum-free medium for the invasion assay. The lower chambers contained medium with 10% FBS. The chambers were incubated for 22 h at 37°C. The cells that invaded into the lower surface of the membranes were fixed, stained, and counted in randomly selected fields under a light microscope. The number of invading CWR22R-1 cells was set as 1, and relative invasiveness was calculated. Columns, mean from three independent experiments; bars, SD.

 
The Antiproliferative Effect of 1,25-VD Is Not Affected by Androgen Signaling
CWR22rv1 is derived from CWR22R, a relapsed tumor after castration-induced regression in the parental CWR22 xenografts (17). Androgen-responsive growth of the two cell sublines was tested and we found that growth of neither CWR22R-1 nor CWR22R-2 is induced by 5{alpha}-dihydrotestosterone (Fig. 3A ). In a previous report, 5{alpha}-dihydrotestosterone stimulated growth of CWR22rv1 in a dose-responsive manner (17). The conflicting results are probably due to the use of serum-free culture medium in the previous study, whereas we used 10% CD-FBS–supplemented medium. Previous reports showed that a mutant androgen receptor protein with a higher molecular weight accompanied by a COOH-terminally truncated androgen receptor species (AR{Delta}LBD) is expressed in CWR22rv1 (18, 19). We observed the same mutant androgen receptor in the two CWR22rv1 sublines used here and Western blot analysis showed the antibody recognizing the NH2 terminus of androgen receptor (NH27), but not the antibody against the COOH terminus of androgen receptor (C-19), detected a truncated form of androgen receptor (AR{Delta}LBD), which was not present in LNCaP cells (Fig. 3B). A previous study showed that Casodex antagonized the antiproliferative effect of 1,25-VD in LNCaP cells, suggesting that 1,25-VD action is androgen dependent in LNCaP (6). To focus on studying the mechanisms contributing to vitamin D resistance besides androgen signaling, which has been well studied, steroids were eliminated from the culture system by using CD-FBS–supplemented medium. This did draw the vitamin D3 response levels of the two sublines closer (from 20% to 10% difference), which suggests vitamin D resistance of CWR22R-2 cells is partially contributed from androgen signaling. Although our study was done in an androgen-depleted culture system, to clarify whether androgen receptor is involved in the antiproliferative effect of 1,25-VD in our system, we used Casodex to antagonize androgen receptor activity. As shown in Fig. 3C, Casodex did not exert a significant effect on the growth response to 1,25-VD in either CWR22R-1 or CWR22R-2, indicating that in this cell model, androgen receptor signaling was not a key factor in the 1,25-VD growth-inhibitory effect under androgen-depleted culture conditions.


Figure 3
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  		FIGURE 3. Characterization of androgen responsiveness and androgen receptor transcriptional activity in CWR22rv1 sublines. A. CWR22R-1 and CWR22R-2 cells were seeded in 24-well plates in 10% CD-FBS–supplemented RPMI. After 24 h, cells were treated with ethanol or 10-9 mol/L 5{alpha}-dihydrotestosterone (DHT). Treatment was re-administered every 2 d. At days 0, 2, 5, and 7, the MTT assay was done. Points, absorbance; bars, SD. B. The expression of androgen receptor was detected in CWR22R-1, CWR22R-2, and LNCaP by Western blotting. Whole-cell lysates harvested from the three cell lines were loaded on a gel and blotted on a membrane. Antibodies against the androgen receptor NH2 terminus (NH27) and COOH terminus (C-19) were used to detect androgen receptor expression. mtAR, mutant androgen receptor; wtAR, wild-type androgen receptor. Different forms of androgen receptor were detected (arrows). C. CWR22R-1 and CWR22R-2 cells were seeded and treated with 100 nmol/L 1,25-VD and 1 µmol/L Casodex alone or together as indicated. After the 6th day of treatment, the MTT assay was done. Each treatment condition was assayed in triplicate, and the percentage of growth inhibition compared with ethanol was calculated. Columns, mean; bars, SD.

 
1,25-VD–Induced VDR Transcriptional Activity Is Reduced in CWR22R-2
The genomic effect of 1,25-VD is mediated through VDR. To examine whether these effects contribute to the differential antiproliferative effect of 1,25-VD, transcriptional activities of VDR in CWR22rv1 sublines were examined and compared by using rCYP24-LUC, a VDR reporter gene. As shown in Fig. 4A , the induction of LUC activities by 1,25-VD was around 10-fold in CWR22R-1, but <3-fold in CWR22R-2. The higher 1,25-VD–induced transcriptional activation in CWR22R-1 was further confirmed by examination of endogenous expression level of CYP24 using semiquantitative reverse transcription-PCR (RT-PCR). After treatment with 1,25-VD for 24 h, CYP24 expression was induced dramatically in CWR22R-1 compared with CWR22R-2 (Fig. 4B). The reduced 1,25-VD–induced CYP24 in CWR22R-2 cells also excluded the possibility of a higher CYP24 activity converting 1,25-VD into less active 1,24,25-dihydroxyvitamin D3 in CWR22R-2 cells. To further support our observations, we examined the expression of protease M, which is another 1,25-VD–regulated gene discovered in squamous carcinoma (20). Similar to CYP24, protease M was found to be induced in CWR22R-2 far less than in CWR22R-1 at 24 h after 1,25-VD treatment (Fig. 4C). Taken together, the transcriptional activity of VDR is weaker in CWR22R-2, which is correlated with reduced growth arrest effects of 1,25-VD in this cell line.


Figure 4
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  		FIGURE 4. The transcriptional activity of VDR between CWR22rv1 sublines. A. CWR22R-1 and CWR22R-2 cells were plated at a density of 105 per well in 24-well plates. VDR transcriptional activity was measured by transfection of 0.5 µg prCYP24-LUC reporter and 1 ng pRL-SV40 plasmids into cells. After 20 h, cells were treated with ethanol or 100 nmol/L 1,25-VD for 24 h before harvesting. The LUC activity relative to lane 1 was calculated. Columns, mean of three independent experiments; bars, SD. B. CWR22R-1 and CWR22R-2 cells were seeded at a density of 106 per 100-mm dish in 10% CD-FBS–supplemented RPMI. After 24 h, cells were treated with 100 nmol/L 1,25-VD for 0, 24, or 72 h before harvesting. Treatment was re-administered every 2 d. Semiquantitative RT-PCR was done to detect the amount of CYP24 in each sample. ß-Actin expression served as an internal control. C. Cells were seeded as previously described and treated with 1,25-VD for 0, 12, 24, or 48 h before harvesting. Semiquantitative RT-PCR was done to detect the expression amount of protease M in each sample. ß-Actin expression served as a control.

 
Expression of 1,25-VD Transporters, VDR, and the Ligand-Binding Affinity of VDR Are Similar in CWR22rv1 Sublines
To further dissect the mechanisms mediating the reduced 1,25-VD response in CWR22R-2 cells, we investigated the impairment of 1,25-VD–induced VDR transcriptional activity. First, we examined the expression of cubilin and megalin that transport 1,25-VD, by using real-time PCR. As shown in Fig. 5A , the expression of cubilin was higher, whereas that of megalin was lower, in CWR22R-1 compared with CWR22R-2 cells. The varying levels of the two transporters between the cell lines suggest that they might compensate for each other's function. Mutations and the expression levels of VDR that could contribute to the transcriptional activity of the receptor were examined next. As shown in Fig. 5B, neither VDR protein level (left) nor VDR mRNA expression (right) was reduced in CWR22R-2. Moreover, we identified no mutation in the VDR cDNAs amplified from the two cell lines (data not shown). Then, the ligand-binding affinity of VDR in these two sublines was examined and Kd values in CWR22R-1 and CWR22R-2 were 0.31 and 0.23 nmol/L, respectively (Fig. 5C). This indicated the lower transcriptional activity in CWR22R-2 was not due to less ligand-binding affinity of VDR. Overall, we found no obvious explanation for the differences in VDR transcriptional activity among the studies of expression of 1,25-VD transporters, or the expression, ligand-binding affinity, or coding sequence of VDR.


Figure 5
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  		FIGURE 5. The expression levels of vitamin D membrane transporters, VDR, and ligand-binding affinity of VDR in CWR22rv1 sublines. A. Reverse transcription was done to amplify cDNA from 4 µg total RNA extracted from CWR22R-1 and CWR22R-2 cells. The mRNA expression of cubilin, megalin, and ß-microglobulin was measured by real-time PCR. After normalization relative to ß-microglobulin expression, the relative amount compared with that of CWR22R-1 (set at 1-fold) was calculated. Columns, mean of triplicate samples; bars, SD. B. Semiquantitative RT-PCR was done to detect the mRNA expression of VDR and ß-actin in each sample. ß-Actin expression served as a control. Sixty micrograms of protein from whole-cell lysates from CWR22R-1 and CWR22R-2 cells were loaded in an 8% gel for detection of VDR and ß-actin by Western blotting. C. Protein extracts were prepared from CWR22R-1 and CWR22R-2 cells and incubated for 16 to 20 h at 4°C in the presence of increasing concentrations of [3H]1,25-VD. Specific binding was measured by subtracting the binding in the presence of a 250-fold excess of radio-inert hormone from total binding. Scatchard plot of the binding data and the calculated ligand-binding affinity.

 
DNA-Binding Ability of VDR Is Decreased in CWR22R-2
1,25-VD signals are mediated by binding of VDR to the VDR response element (VDRE) in the promoters of target genes and turning on transcription. Therefore, we compared VDR DNA-binding capacity in the two CWR22rv1 sublines. For electrophoretic mobility shift assay (EMSA), nuclear extracts containing similar amounts of VDR from the two cell lines were prepared (Fig. 6A, top ) and incubated with isotope-labeled oligonucleotides containing VDRE. As shown in Fig. 6A (bottom), VDR-VDRE complexes formed, as indicated by arrows, consisting of two groups that have been suggested to be VDR-RXR heterodimers bound VDRE and VDR homodimers bound VDRE (21). These VDR-VDRE complexes were attenuated by an antibody (9A7) against VDR. Less VDR-VDRE complexes were formed in CWR22R-2 nuclear extracts compared with CWR22R-1 nuclear extracts (Fig. 6A, bottom). No significant change was observed when 1,25-VD was added into the EMSA reaction. To verify this result, we did a DNA pull-down assay in which oligonucleotides containing VDRE were applied to pull down VDRE-binding protein complexes from nuclear extracts. VDR presented in the pull-down complexes was then detected by Western blotting. As shown in Fig. 6B (bottom), the results showed that less VDR-VDRE existed in the DNA pull-down complexes from CWR22R-2 nuclear extracts, although they contained more VDR (Fig. 6B, top). Both EMSA and DNA pull-down assays suggest the DNA-binding ability of VDR is weaker in CWR22R-2 than that in CWR22R-1. Furthermore, we did a chromatin immunoprecipitation (ChIP) assay to measure the in vivo VDR-VDRE complex in CYP24 promoter. To our surprise, higher amounts of VDR-VDRE complex existed in the CYP24 promoter in CWR22R-2 cells compared with CWR22R-1 (Fig. 6C, IP with {alpha}VDR). Upon treatment with 1,25-VD, VDR-VDRE complex increased, whereas NCoR1 in VDRE decreased, in both cell lines (Fig. 6C, IP with {alpha}VDR and {alpha}NCoR1, lanes 4 and 2). We also observed that a relatively higher amount of NCoR1 existed in the VDRE of CWR22R-2 cells compared with CWR22R-1 cells (Fig. 6C, IP with {alpha}NCoR1). These results suggest the presence of NCoR1 exerts a transrepression effect to counteract VDR-activating target gene expression.


Figure 6
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  		FIGURE 6. The DNA-binding ability of VDR in CWR22rv1 sublines in EMSA, DNA pull down, and ChIP assays. A. CWR22R-1 and CWR22R-2 cells were seeded in 100-mm dishes, in 10% FBS-supplemented RPMI, for 2 d. Medium was changed to 10% CD-FBS–supplemented RPMI the day before treating with ethanol or 100 nmol/L 1,25-VD. After 2-h treatment, nuclear extracts (NE) were prepared from each sample. Top, the VDR input detected by Western blotting. Ten micrograms of nuclear extracts were incubated with an oligonucleotide probe end-labeled with [{gamma}-32P]ATP in EMSA buffer for 15 min at room temperature. Antibody or control IgG was then added to the mixture and incubated for another 15 min. Bottom, the protein-DNA complexes analyzed on a 6% native polyacrylamide gel. B. CWR22R-1 and CWR22R-2 cells were seeded and treated as described in A. Two hundred micrograms of nuclear extracts were incubated with oligonucleotide-conjugated beads in EMSA buffer for 1 h at 4°C. The mixture was centrifuged and washed thrice with 0.5% Tween 20/PBS. Top, 10% of VDR input detected by Western blotting. Bottom, VDR in pull-down protein-DNA complex detected by Western blotting. C. Cells were seeded and treated as described in A, then subjected to ChIP assay. Sonicated nuclear lysates were immunoprecipitated (IP) with IgG, {alpha}VDR, or {alpha}NCoR1 antibody. The CYP24 VDRE contained in the DNA fragment purified from the pull-down lysates were amplified by PCR and visualized in 2% agarose gel.

 
The Expression of RXR and Function of RXRß Are Normal, whereas the Expression of Corepressors Is Higher in CWR22R-2
We hypothesize the profile of interacting proteins of VDR might be different between CWR22R-1 and CWR22R-2, therefore contributing to less VDR-VDRE in CWR22R-2. Earlier reports showed maximum or proper transcriptional activity of VDR requires interaction with RXR (22). As shown in Fig. 7 , we found that the RNA levels of three RXR isoforms are variable between the two cell lines, but not always lower in CWR22R-2 (Fig. 7A). Therefore, the reduction of DNA-binding ability of VDR in CWR22R-2 may not be due to insufficient amounts of RXR. We then analyzed the formation of VDR-RXRß heterodimers to investigate the function of RXRß in these two cell lines and found no difference of functional RXRß (Fig. 7B, lanes 4 and 5). Next, we examined the expression profiles of coregulators in these two sublines by using real-time PCR. Among all the coregulators examined, we found that the expression of two corepressors, NCoR1 and SMRT, was higher in CWR22R-2 (Fig. 7C). Higher expression of corepressors NCoR1 and SMRT could contribute to the lower transcriptional activity of VDR in CWR22R-2. In summary, we found that CWR22R-2, more aggressive prostate cancer cells, expressed higher amounts of corepressors NCoR1 and SMRT than CWR22R-1, which resulted in less available VDR binding to VDRE and reduced VDR transcriptional activity, thus leading to decreased antiproliferative effect of 1,25-VD.


Figure 7
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  		FIGURE 7. The expression and function of RXRß and the expression of corepressors in CWR22rv1 sublines. A. Reverse transcription was done to amplify cDNA from 4 µg total RNA extracted from CWR22R-1 and CWR22R-2 cells. The expression of RXR isoforms and ß-microglobulin was measured by real-time PCR. After normalization relative to ß-microglobulin expression, gene expression in CWR22R-2 compared with CWR22R-1 was calculated. Columns, mean of triplicate samples; bars, SD. B. The GST pull-down assay was applied to detect VDR interacting with RXRß in CWR22R-1 and CWR22R-2 cells. GST-VDR-L was purified by incubating with glutathione-conjugated beads. After incubating GST-VDR-L with ethanol or 1 µmol/L 1,25-VD for 2 h, 2 mg nuclear extract from cells was added and incubated for another 2 h. The pull-down complex was loaded on an 8% gel, and RXRß was detected by Western blotting. C. The expression of VDR coregulators and ß-microglobulin was measured by real-time PCR as described in A. After normalization relative to ß-microglobulin expression, the expression of NCoR1 and SMRT in CWR22R-2 relative to CWR22R-1 was calculated. Columns, mean of triplicate samples; bars, SD.

 
Histone Deacetylase Inhibitor Promotes VDR Transcriptional Activity and the Antiproliferative Effect of 1,25-VD
Higher expression of these two corepressors implied that histone deacetylase activity is increased because they are components of histone deacetylase (23). To test whether enhancement of VDR transcriptional activity in CWR22R-2 cells can restore the antiproliferative effect, trichostatin A (TSA), a histone deacetylase inhibitor was applied to modulate VDR transactivation. First, we showed that the 1,25-VD–induced VDR activity was increased by TSA (Fig. 8A, lanes 2 and 3 ). Also with EB 1089, a synthetic vitamin D analogue, induction of VDR transcriptional activity was potentiated by cotreatment with TSA (Fig. 8A, lanes 4 and 5). Next, we tested whether increased VDR transcriptional activity by TSA also promoted the antiproliferative effect of 1,25-VD and EB 1089. TSA alone exerts 15% growth inhibition (Fig. 8B, lane 1). In combination, TSA and 1,25-VD have no significant additive effect (Fig. 8B, lane 3 versus lane 2). This may be due to the higher turnover rate of 1,25-VD, resulting from increased CYP24 expression after TSA enhancement of VDR activity. On the other hand, the antiproliferative effect of EB 1089, which is not metabolized by CYP24 (20), was further potentiated with addition of TSA (Fig. 8B, lane 5 versus lane 4). Together, we showed that the antiproliferative effect of EB 1089 and the EB 1089–induced VDR transcriptional activity are correspondingly enhanced by TSA in CWR22R-2 cells. This suggests that enhancement of VDR transcriptional activity restores antiproliferation effects of 1,25-VD in CWR22R-2 cells.


Figure 8
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  		FIGURE 8. The influence of histone deacetylase inhibitor TSA on VDR transcriptional activity and on antiproliferative effects of 1,25-VD. A. CWR22R-2 cells were plated at a density of 105 per well in 24-well plates. VDR transcriptional activity was measured by transfection of 0.5 µg prCYP24-LUC reporter and 1 ng pRL-SV40 plasmids into cells. After 20 h, cells were treated with ethanol, 100 nmol/L 1,25-VD, or 10 nmol/L EB 1089 alone or in combination with 10 nmol/L TSA as indicated, for 24 h before harvesting. The LUC activity relative to the ethanol-treated sample was calculated. Columns, mean of three independent experiments; bars, SD. B. CWR22R-2 cells were seeded at a density of 5 x 104 per well in 24-well plates and cultured in 10% CD-FBS supplemented RPMI. After 24 h, cells were treated with ethanol, 100 nmol/L 1,25-VD, or 10 nmol/L EB 1089, alone or in combination with 10 nmol/L TSA, for 4 d. On day 4, the MTT assay was done to measure cell growth inhibition. The percentage of growth inhibition attained by comparing with ethanol treatment was calculated. Columns, mean of triplicate samples; bars, SD.

 
NCoR1 and SMRT Are Critical for Growth-Inhibition Effect of 1,25-VD
To investigate whether the higher expression levels of NCoR1 and SMRT in CWR22R-2 result in resistance to 1,25-VD, we designed small interfering RNAs (siRNA) targeting NCoR1 and SMRT to knock down their expression. CWR22R-2 clones that stably express scrambled control siRNA (SC10), NCoR1 siRNA (N1-2), and SMRT siRNA (S2) were established by retrovirus infection. We first measured the expression level of targeted genes in these two clones. The RNA expression levels measured by real-time PCR showed around 40% down-regulation of NCoR1 in the N1-2 clone and 53% down-regulation of SMRT in the S2 clone compared with the SC10 clone (Fig. 9A ). We next measured the growth inhibition effects of 1,25-VD in these clones. SC10 cells treated with 100 nmol/L 1,25-VD for 6 days showed 8% growth inhibition, whereas N1-2 showed 15% and S2 showed 16% growth inhibition (Fig. 9B). Furthermore, the transactivity of VDR was examined by reporter gene assay. In N1-2 and S2 cells, 1,25-VD treatment induced LUC activity 5-fold, whereas induction was only 3-fold in SC10 cells (Fig. 9C). Although the transactivity of VDR was recovered, it was not completely restored to the same level as in CWR22R-1. This suggests that other factors also contribute to the vitamin D resistance and determining these factors required future studies. Overall, targeted down-regulation of NCoR1 or SMRT can partially restore 1,25-VD sensitivity in CWR22R-2 cells. This result indicates the higher expression levels of NCoR1 and SMRT interfere with 1,25-VD/VDR–mediated growth inhibition in the CWR22R-2 cell line.


Figure 9
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  		FIGURE 9. Down-regulation of NCoR1 and SMRT by siRNA sensitize CWR22R-2 to 1,25-VD–induced growth inhibition. A. CWR22R-2 clones that stably express scrambled control siRNA (SC10), NCoR1 siRNA (N1-2), or SMRT siRNA (S2) were established by retrovirus infection. RNA expression level of NCoR1 in N1-2 and SMRT in S2 compared with SC10 cells were measured by real-time PCR. B. SC10, N1-2, and S2 were seeded at a density of 5 x 104 per well in 24-well plates and cultured in 10% CD-FBS–supplemented RPMI. After 24 h, cells were treated with ethanol or 100 nmol/L 1,25-VD. Treatment was refreshed every 2 d. After treating for 6 d, MTT assay was done to measure cell growth–inhibition effect. The percentage of growth inhibition attained by comparing with ethanol treatment was calculated. Columns, mean of triplicate samples; bars, SD. *, P < 0.05 compared with SC10. C. The transactivity of VDR in SC10, N1-2, and S2 cells were assayed as described in Fig. 4A. The LUC activity relative to ethanol treatment of each cell line was calculated. Columns, mean of three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01 compared with SC10.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
To study the mechanisms controlling the development of 1,25-VD resistance in prostate cancer cells, we compared the antiproliferative effects of 1,25-VD in several established prostate cancer cell lines and selected the CWR22rv-1 sublines, CWR22R-1 and CWR22R-2, as our experimental models. Both cell lines are androgen independent and express a mutant androgen receptor, as does the parental line CWR22rv-1. The faster growth rate and more pronounced invasive ability of CWR22R-2 compared with CWR22R-1 cells indicate that the CWR22R-2 line is more aggressive than the CWR22R-1 line. By studying these two sublines, we found that the transcriptional activity of VDR, but not its expression level, is lower in CWR22R-2. Also, modulation of the transcriptional activity of VDR by a histone deacetylase inhibitor promotes the 1,25-VD responsiveness in CWR22R-2. Down-regulation of NCoR1 and SMRT, two corepressors with higher expression levels in CWR22R-2, sensitize CWR22R-2 to the growth inhibition effects of 1,25-VD. Therefore, the transcriptional activity of VDR plays an important role in the antiproliferative effect of 1,25-VD. This is the first study showing differential antiproliferative effects of 1,25-VD in a pair of sublines derived from one parental prostate cancer cell line.

In addition to studies of prostate cancer, there have been several studies in the breast cancer field using vitamin D–resistant variants of MCF-7 as models to investigate the underlying mechanisms of vitamin D resistance (24, 25). Although differences in expression or transcriptional activity of VDR were not found, 12-O-tetradecanoylphorbol-13-acetate pretreatment of MCF-7D3Res cells could enhance 1,25-VD–stimulated transcriptional activity and VDR protein expression, and sensitize cells to respond to 1,25-VD (26). In addition to breast cancer cell lines, transformed mammary cells isolated from VDR knockout mice showed no response to 1,25-VD, whereas growth of cells from wild-type mice was inhibited (27). Vitamin D resistance had also been shown in many other cell types, and numerous mechanisms have been found. In a vitamin D–resistant variant of the human myeloid leukemia cell line HL-60, reduced amounts of the specific cytosolic receptor for vitamin D were observed (28). In osteosarcoma cells, functional VDRs and RXRs were found, but the degradation of RXR results in resistance to vitamin D treatment (22). A patient with resistance to vitamin D has normal VDR expression; however, VDR transactivation was suppressed by overexpression of hnRNPA1, a homologue of the VDRE-binding protein found in primates, which blocks the DNA-binding ability of VDR (29). Several mutations in the VDR gene have been shown to cause hereditary vitamin D–resistant rickets (30-32). Overall, the expression and transcriptional activity of functional VDR, and proteins involved in VDR function, are important in determining the response to vitamin D in several types of cells.

Prompted by our finding showing weaker transcriptional activity of VDR in CWR22R-2 cells, we further investigated the mechanism of this effect from several directions, as summarized in Fig. 10 , including mechanisms that have and have not been addressed in previous reports. First, mutations of the VDR cDNA were not found in CWR22R-2. Second, expression of RXRß and the formation of VDR-RXRß heterodimers were normal. Third, overexpression of hnRNPA1 and B1 were not found in CWR22R-2 based on quantification of mRNA levels of these genes by real-time PCR (data not shown). In addition to the known mechanisms controlling 1,25-VD responsiveness described in previous reports, we also explored some that had not been studied in other vitamin D–resistant cells, but that are also important for VDR function. 1,25-VD transporter protein expression and the ligand-binding affinity of VDR both appeared to be normal. Intriguingly, we did find that the DNA-binding ability of VDR was weaker in CWR22R-2 using in vitro assay. However, more VDR-VDRE complex were found in CWR22R-2, where more NCoR1 also resided in VDRE. Examination of VDR coregulator expression indicated that higher expression of the corepressors NCoR1 and SMRT in CWR22R-2 might contribute to suppression of VDR transcriptional activity.


Figure 10
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  		FIGURE 10. Diagram of VDR-mediated growth inhibition in response to 1,25-VD. 1,25-VD enters cells via diffusion or transporter proteins, cubilin, and megalin, and binds to VDR. VDR without ligand is associated with corepressors (NCoR), such as NCoR1 and SMRT, which dissociate from VDR after ligand binding. Ligand-bound VDR forms a heterodimer with RXR and recruits coactivators (NCoA), such as SRC-1, p300, etc., resulting in histone acetylation (Ac). The DRIP complex and transcriptional machinery join VDR to initiate transcription of accessible DNA templates. The expression of VDR target genes results in cell growth inhibition.

 
Although we observed no mutations in the VDR coding sequence in either CWR22rv1 subline, it is possible that posttranslational modification of VDR protein affects transcriptional activity. There is evidence showing acetylation to be one type of posttranslational modification of nuclear receptors (33, 34). One report showed that TSA synergistically cooperated with 1,25-VD in growth inhibition of prostate cancer cells (35). Based on our observation that TSA can promote 1,25-VD–induced VDR transcriptional activity and growth inhibition, it is possible that transactivation of VDR can be modulated directly by acetylation. On the other hand, increased expression of corepressors SMRT and NCoR1, which are components of the histone deacetylase complex (23) and are activation cofactors for histone deacetylases (36), might suppress VDR transcriptional activity in CWR22R-2. TSA might therefore indirectly modulate VDR transcriptional activity by attenuating the effects of the corepressors. Further investigation of this hypothesis by siRNA targeting down-regulation of NCoR1 and SMRT indeed showed that these two corepressors interfere with 1,25-VD induced growth inhibition. A recent study suggests that SMRT expression is frequently elevated in malignant prostate cells and is often associated with reduced sensitivity to 1,25-VD, and that siRNA targeted down-regulation of SMRT did enhance the 1,25-VD–induced gene expression (37). Therefore, the expression levels of NCoR1 and SMRT might serve as markers for predicting sensitivity to vitamin D treatment in prostate cancer.

Here, we provide a pair of prostate cancer sublines, CWR22R-1 and CWR22R-2, derived from CWR22rv1, as a model for studying the mechanisms of vitamin D resistance. The more aggressive character of CWR22R-2 and its reduced response to vitamin D indicate that the effects of vitamin D treatment might be lost during cancer progression. Increasing numbers of studies have been carried out and various mechanisms of vitamin D resistance have been identified which support defects in genomic 1,25-VD/VDR signaling as the major cause. In our model, the DNA-binding ability of VDR is weakened and may result in reduced VDR transcriptional activity. Further research will help clarify the cause of the loss of the DNA-binding ability of VDR, which might involve posttranslational modifications, altered coregulator expression, or other yet unknown mechanisms. By compensating for lost signaling pathways or by blocking the amplification of signals interfering with VDR transcriptional activity, we envision the ability to reestablish responsiveness to vitamin D in prostate cancer cells. Further insight regarding VDR function will be beneficial for therapeutic designs using vitamin D to fight prostate cancer progression.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Plasmids
The cDNA encoding the ligand-binding domain of VDR (VDR-L) was amplified by PCR and inserted into the pGEX-KG vector to generate the glutathione S-transferase (GST)–VDR-L expression plasmid. prCYP24-luciferase (LUC) was constructed by inserting the fragment containing the –950 to –55 bp region of rCYP24 promoter released from rat 25-hydroxyvitamin D3 24-hydroxylase gene promoter construct (–2.2 kb to +188 bp), a generous gift from Dr. Yoshihiko Ohyama (Hiroshima University, Hiroshima, Japan), by MscI and SacI into SacI and SmaI sites of pGL3-TK (a gift from Dr. Eungseok Kim, University of Rochester, Rochester, NY).

Materials
1,25-VD and EB 1089 were generous gifts of Dr. Lise Binderup (Leo Pharmaceutical Products, Ballerup, Denmark). 5{alpha}-Dihydrotestosterone was obtained from Sigma. The VDR antibody for EMSA was purchased from Chemicon; for Western blotting, the antibody was from Santa Cruz Biotechnology. The antibody against ß-actin was purchased from Santa Cruz Biotechnology; the antibody against RXRß was purchased from Affinity Bioreagents; anti-androgen receptor polyclonal antibody, NH27, was a gift from Dr. Chawnshang Chang (University of Rochester, Rochester, NY); and the anti-androgen receptor antibody, C19, was purchased from Santa Cruz Biotechnology.

Cell Culture, Transfection, and Luciferase Assays
CWR22R-1 and CWR22R-2 were gifts from Drs. Ching-Hai Kao (Indiana University, Indianapolis, IN) and Franky L. Chan (The Chinese University of Hong Kong, Hong Kong, China), respectively. Cells were maintained in 10% FBS supplemented RPMI 1640 (Life Technologies) containing penicillin (100 IU/mL) and streptomycin (100 mg/mL). Transfections were done by using SuperFect according to the manufacturer's suggested procedures (Qiagen). After transfection, cells were treated with either ethanol or ligands in medium containing 10% CD-FBS for 24 h. Cell lysates were prepared and the LUC activity was normalized for transfection efficiency using pRL-SV40 as an internal control. LUC assays were done using the dual-LUC reporter system (Promega).

Cell Proliferation Assay
Cells were seeded in 24-well tissue culture plates in RPMI 1640 containing 10% CD-FBS. After incubation for 24 h, the medium was replaced with fresh medium containing 10% CD-FBS and treated with ethanol (final concentration 0.1%), 1,25-VD, or 5{alpha}-dihydrotestosterone at indicated concentrations. At the indicated time points, medium was replenished and cell proliferation was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay according to the manufacturer's manual (Sigma). Stock solution of MTT (5 mg/mL PBS) was added into each well to a 10-fold dilution. After 4-h incubation at 37°C, the stop solution was added to extract the formazan product and the absorbance was recorded. Data are expressed as the mean ± SD of triplicate samples.

Flow Cytometric Analysis
After indicated treatments, cells were harvested and fixed in 70% ethanol overnight. Cells were then pelleted and incubated in PBS containing 0.05 mg/mL RNase A for 30 min at room temperature. After washing, the cells were stained with 10 µg/mL propidium iodide. Cell cycle profiles were determined by using the FACScan flow cytometer, and cell cycle analyses of DNA histograms were done with ModFitLT software.

Invasion Assay
CWR22R-1 and CWR22R-2 cells were harvested and counted, and 105 cells per upper chamber were used for each invasion assay. Cells were added to Matrigel-coated inserts (Becton Dickinson Labware) in serum-free medium. The lower chambers contain medium with 10% FBS. The chambers were incubated for 22 h at 37°C. The cells that invaded into the surface of the membranes were fixed and stained with 1% toluidine blue, and random fields were counted under a light microscope.

SNP Array Genotyping
Genome-wide SNP analysis of the CWR22R-1 and CWR22R-2 cell lines was done on the Affymetrix GeneChip Human Mapping 10 K Array. These arrays contain 10,204 unique SNPs with the median physical distance between adjacent SNPs of 105 kb. Heterozygosity for the array averages 0.37. Each SNP is represented by sense and antisense oligonucleotides for each SNP variant as well as single base pair mismatches. Genomic DNA was extracted from each cell line using Qiagen Qiamp DNA Mini Kit according to the manufacturer's directions. Genomic DNA was digested with XbaI and then ligated to adapter oligonucleotides, which serve as priming sites for sequence independent PCR amplification. For quality control, an aliquot of the PCR reaction was analyzed by gel electrophoresis. Three distinct bands from repetitive sequence DNA and a smear from single-copy DNA were anticipated and were observed to confirm the successful amplification of sample DNA. Following fragmentation with DNase I and end labeling with biotin, the DNA was hybridized to the SNP array and detected with fluorescently labeled avidin. Using Affymetrix GeneChip DNA Analysis Software, each locus was assigned one of three genotypes, AA or BB homozygous, or AB heterozygous. Ambiguous values received a "no call" assignment.

Semiquantitative RT-PCR and Real-time PCR Assay
Total RNA was extracted from cells using TRIzol (Invitrogen). Semiquantitative RT-PCR was carried out by reverse transcription with the SuperScript II kit (Invitrogen) and PCR amplifications with primers for VDR and CYP24 (38). Primers for ß-actin were sense 5'-TGTGCCCATCTACGAGGGGTATGC-3' and antisense 5'-GGTACATGGTGGTGCCGCCAGACA-3'. The PCR was done as follows: initial denaturation at 95°C for 10 min, and 35 cycles (CYP24) or 16 cycles (VDR and ß-actin) of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. Real-time PCR was done with SYBR Green PCR Master Mix on an iCycler IQ multicolor real-time PCR detection system (Bio-Rad). Primer sequences were ß-microglobulin: sense 5'-GATGAGTATGCCTGCCGTGT-3', antisense 5'-AATTCATCCAATCCAAATGAG-3'; cubilin: sense 5'-ATTGCTCCAGGTGCTTGTG-3', antisense 5'-GTTGAGTTCCACGGTAGAGTC-3'; megalin: sense 5'-TCGGAAGAGTGGAAGTGTGATAATG-3', antisense 5'-AGGTGAAGGCTGTCGGTGAG-3'; RXR{alpha}: sense 5'-TTCGCTAAGCTCTTGCTC-3', antisense 5'-ATAAGGAAGGTGTCAATGGG-3'; RXRß: sense 5'-GAAGCTCAGGCAAACACTAC-3', antisense 5'-TGCAGTCTTTGTTGTCCC-3'; RXR{gamma}: sense 5'-GCAGTTCAGAGGACATCAAGCC-3', antisense 5'-GCCTCACTCTCAGCTCGCTCTC-3'; NCoR1: sense 5'-CGTTACAACACTGCTGCGGATG-3', antisense 5'-ACTGCTGCTGACCTGCTTCTC-3'; SMRT: sense 5'-GGTGGTGGTGAGGACGGTATTG-3', antisense 5'-GGCGGCTGGCTGGTGTTG-3'. The PCR was done as follows: initial denaturation at 95°C for 10 min, and 45 cycles of denaturation at 95°C for 30 s, annealing at 65°C for 30 s, and extension at 72°C for 30 s. By subtracting the threshold cycle value (CT) from the corresponding ß-microglobulin CT value (internal control) from each time point, {Delta}CT values were calculated. Then, relative amounts were calculated by comparing the {Delta}CT value of CWR22R-2 to the {Delta}CT value of CWR22R-1 samples.

Electrophoretic Mobility Shift Assay
The VDRE probe was from the human CYP24 gene. The oligonucleotide probes for EMSA were end-labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase (New England BioLabs). CWR22R-1 and CWR22R-2 nuclear extracts were prepared according to procedure described previously (39). The EMSA reactions were done with 10 µg of nuclear extracts in an EMSA buffer containing 10 mmol/L Tris (pH 7.4), 4% (v/v) glycerol, 100 mmol/L KCl, 0.5 mmol/L, 2 µg/µL bovine serum albumin, and 1 mmol/L DTT. The reaction mixtures of the proteins and DNA were incubated for 15 min at room temperature. For the antibody supershift assay, the mixtures were incubated for another 15 min in the presence or absence of VDR antibody (9A7; Chemicon). The protein-DNA complexes were analyzed on a 6% native polyacrylamide gel.

DNA Pull-Down Assay
Oligonucleotides corresponding to the hCYP24 VDRE were synthesized as follows: biotin-VDRE1(hCYP24), 5'-biotin-GATCGGAGTTCACCGGGTGTG-3'; biotin-VDRE2(hCYP24), 5'-biotin-GATCCACACCCGGTGAACTCC-3'. Double-stranded probes were made by annealing 100 ng/µL mixture of complimentary oligonucleotides in TNE [10 mmol/L Tris-Cl, 50 mmol/L NaCl, and 1 mmol/L EDTA (pH 8.0)] by heating to 95°C for 5 min followed by slowly cooling to room temperature. Double-stranded probes were then immobilized on streptavidin-agarose by incubating in 0.2% bovine serum albumin/PBS at 4°C for 1 h and then washed thrice with PBS. Nuclear extract (200 µg) from CWR22R-1 and CWR22R-2 cells were prepared and incubated in binding buffer [10 mmol/L Tris-HCl, 100 mmol/L KCl, 1 mmol/L DTT, 15% glycerol, 0.2% bovine serum albumin and 25 µg/mL poly(deoxyinosinic-deoxycytidylic acid)] with or without immobilized probe on streptavidin agarose at 4°C for 2 h. The complexes were collected by pulse centrifugation in a microcentrifuge, washed thrice with PBS containing 0.05% Tween 20, eluted by addition of sample buffer, and boiled for 5 min. Proteins were then separated by 10% SDS-PAGE and analyzed by immunoblot.

Chromatin Immunoprecipitation Assay
ChIP assay was carried out using the Upstate Biotechnology ChIP assay kit with modifications. In brief, cells treated with ethanol or 1,25-VD were cross-linked with 1% formaldehyde, lysed, and chromatin pellets were sonicated to an average of 200- to 1,000-bp fragments of DNA. The chromatin fragments were subjected to immunoprecipitation with 2 µg VDR antibody (H-18 from Santa Cruz Biotechnology) overnight at 4°C. The precipitates were eluted by the elution buffer (1% SDS, 100 mmol/L NaHCO3, and 10 mmol/L DTT). The cross-links were reversed with 4-h incubation at 65°C in the elution buffer with addition of 200 mmol/L NaCl. The immunoprecipitated DNA fragments were purified using Qiagen MiniElute Reaction Cleanup kits and subjected to PCR using a pair of primers (sense 5'-CGCCCAGCGAACATAGCC-3' and antisense 5'-CCAATGAGCACGCAGAGG-3'), which were designed to amplify the VDRE in CYP24 promoter.

GST Pull-Down Assay
GST-VDR-L fusion proteins and GST control protein were purified (40). The GST fusion proteins were pulled down by glutathione beads at 4°C for 1 h and washed thrice with washing buffer. The purified GST fusion proteins and beads were incubated in 100 µL binding buffer with ethanol or 1 µmol/L 1,25-VD for 1 h, at 4°C. Nuclear extracts prepared from cells were added and incubated for another 2 h at 4°C. Glutathione beads were washed with washing buffer thrice. Protein complexes were separated by 10% SDS-PAGE and RXRß was detected by Western blotting.

Western Blotting
Total cell lysates were prepared by lysing cells in ice-cold radioimmunoprecipitation assay buffer (1% Igepal CA-630, 0.5% sodium deoxycholate, and 0.1% SDS in PBS). The lysates were clarified by centrifugation. The protein concentrations of the supernatants was evaluated with the Bio-Rad reagent kit. From each sample, 60 µg protein was separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in TBST [10 mmol/L Tris-Cl (pH 7.4), 150 mmol/L NaCl, 0.05% Tween 20] containing 5% nonfat dry milk for 1 h at room temperature. Primary antibodies in TBST were added and incubated at 4°C overnight, and then the alkaline phosphatase–conjugated secondary antibodies (Santa Cruz Biotechnology) in TBS were added and incubated for 1 h at room temperature. The membranes were washed thrice in TBST (10 min at room temperature) and the immunoreactive bands were visualized by alkaline phosphatase activity with the 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium phosphatase substrate (Bio-Rad Laboratories).

Construction of DNA Vector–Based RNA Interference Plasmids and Stable Cell Lines
Small interfering RNA target sites were selected by using the siRNA design tool on Ambion website and OligoEngine Workstation 2 software. The corresponding oligonucleotides for generating the scrambled control siRNA insert are 5'-GATCCCCAACAAGCACACACGTGTCTTTCAAGAGAAGACACGTGTGTGCTTGTTTTTTTA-3' (forward) and 5'-AGCTTAAAAAAACAAGCACACACGTGTCTTCTCTTGAAAGACACGTGTGTGCTTGTTGGG-3' (reverse). The corresponding oligonucleotides for generating the NCoR1 siRNA insert are 5'-CGCGGGCCCTGGGCAGCAATTGCTAAACAAGAGTTTA-3' (forward) and 5'-CGGAATTCAAAAACTGGGCAGCAATTGCTAAACTCTTG-3' (reverse). The corresponding oligonucleotides for generating the SMRT siRNA insert are 5'-GATCCCCGGTCCAACACACTTGACAATTCAAGAGATTGTCAAGTGTGTTGGACCTTTTTA-3' (forward) and 5'-AGCTTAAAAAGGTCCAACACACTTGACAATCTCTTGAATTGTCAAGTGTGTTGGACCGGG-3' (reverse). These were subsequently cloned into the pMSCV/U6 (41) or pSuperior.retro.puro (OligoEngine). Stable cell lines expressing these siRNAs were generated according to manufacturer's manual (OligoEngine).


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Drs. Yoshihiko Ohyama, Eungseok Kim, Lise Binderup, Chawnshang Chang, Ching-Hai Kao, and Franky L. Chan for kindly providing plasmids, reagents, antibodies, and cell lines for this study.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: Department of Defense grant PC040630.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/26/06; revised 5/21/07; accepted 6/ 5/07.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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