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Endocrinology Vol. 144, No. 1 50-60
Copyright © 2003 by The Endocrine Society

ARTICLE

p53 Is Required for 1,25-Dihydroxyvitamin D3-Induced G0 Arrest But Is Not Required for G1 Accumulation or Apoptosis of LNCaP Prostate Cancer Cells

Tara C. Polek, LaMonica V. Stewart, Elizabeth J. Ryu, Michael B. Cohen, Elizabeth A. Allegretto and Nancy L. Weigel

Department of Molecular and Cellular Biology (T.C.P., L.V.S., E.J.R., N.L.W.), Baylor College of Medicine, Houston, Texas 77030; Department of Pathology, Urology, and Epidemiology (M.B.C), University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa 52242; and Ligand Pharmaceuticals, Inc. (E.A.A.), San Diego, California 92121

Address all correspondence and requests for reprints to: Dr. Nancy L. Weigel, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: nweigel{at}bcm.tmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is an effective agent for inhibiting the growth of prostate cancer cells including LNCaP and PC-3 cell lines. However, the extent of growth inhibition in these cell lines differs because LNCaP cells are much more responsive than PC-3 cells. Previous studies in LNCaP cells have shown that 1,25-(OH)2D3 treatment results in G0/G1 cell cycle accumulation, loss of Ki67 expression, and induction of apoptosis. One difference between the two cell lines is that PC-3 cells lack functional p53, a protein that plays roles both in cell cycle regulation and induction of apoptosis. In this study, the role of p53 in 1,25-(OH)2D3 action was examined using the p53-negative PC-3 cells and a line of LNCaP cells, called LN-56, in which p53 function was shut off using a dominant negative p53 fragment. We found that treatment with 1,25-(OH)2D3 extensively inhibits growth of LN-56 prostate cancer cells lacking p53, but in contrast to the parental LNCaP cells, the LN-56 cells recover rapidly. Moreover, in prostate cancer cells, the synergism between 1,25-(OH)2D3 and 9-cis retinoic acid appears to be dependent on the presence of functional p53; however, 1,25-(OH)2D3-mediated induction of G1 cell cycle accumulation and induction of apoptosis is not.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE ARE LIMITED options for the treatment of prostate cancer once a tumor has escaped the confines of the prostate. Although most metastatic prostate cancers are initially responsive to androgen ablation therapy, these tumors eventually become androgen independent, necessitating a search for alternative treatments. One potential alternative is the use of analogs of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the active metabolite of vitamin D. 1,25-(OH)2D3 treatment inhibits the growth of multiple types of cancer cells including breast (1, 2), colon (3), and prostate cancer cell lines (4, 5, 6, 7). These growth-inhibitory effects of 1,25-(OH)2D3 are mediated via the vitamin D receptor (VDR), a ligand-activated transcription factor, which is a member of the steroid/thyroid nuclear receptor superfamily (8). VDR is expressed in most prostate cancer cell lines (7), including the three best characterized human prostate cancer cell lines (LNCaP, PC-3, and DU145 cells) as well as in primary cultures of prostatic cells (4, 9, 10).

Cellular responsiveness to 1,25-(OH)2D3 varies among prostate cancer cell lines. Although the LNCaP cells are extensively growth inhibited by 1,25-(OH)2D3, PC-3 cells are much less responsive and DU145 cells are virtually nonresponsive to 1,25-(OH)2D3 treatment (4). The differences in the response of the three cell lines cannot be attributed solely to the relative levels of VDR. The nonresponsive DU145 cells express functional VDR (4, 7) and stable transfection of PC-3 cells with an expression plasmid to increase VDR levels does not enhance their responsiveness to 1,25-(OH)2D3 (11).

Because of their independent isolation, there are many differences among the three cell lines. One difference is that neither the PC-3 nor DU145 cells express androgen receptor (12). However, complete loss of androgen receptor expression in prostate cancers is rare (13). Another difference is that LNCaP cells express wild-type, functional p53, whereas PC-3 and DU145 cells do not (14, 15). Although p53 mutations are rare in early stages of prostate cancer (16, 17, 18), they are more common in late stages of the disease [reports vary between 56% and 71% of metastatic prostate cancers with p53 mutations (19, 20, 21)]; therefore, it is important to determine whether p53 plays a role in 1,25-(OH)2D3-mediated growth inhibition of prostate cancer cells.

The tumor suppressor gene p53 is the most frequently mutated gene in human cancer (22, 23). This high-mutation frequency is attributed to the fact that p53 is a critical regulator of the G1/S cell cycle checkpoint as well as being important in many apoptotic pathways (24, 25). Thus, cancer cells with mutations in p53 or loss of p53 expression gain a growth advantage because they can escape the critical G1 cell cycle checkpoint that normally guards against division of cells with DNA damage, and they are less susceptible to removal by apoptosis. Regulation of cell cycle and apoptotic pathways by p53 occurs in part through transcriptional regulation of its target genes. This leads to increases in protein levels of cell cycle inhibitors and apoptosis promoters such as p21 and bax, respectively, or decreases in protein levels of survival factors such as Bcl-2 (25, 26).

Our studies and those of others examining the mechanism of 1,25-(OH)2D3 action in LNCaP cells have shown that 1,25-(OH)2D3 arrests LNCaP cells in the G0/G1 phase of the cell cycle (27, 28, 29) and induces apoptosis (29, 30, 31). G1 arrest of LNCaP prostate cancer cells induced by 1,25-(OH)2D3 is accompanied by an induction of the cyclin-dependent kinase inhibitor p21 (27), and apoptosis induced by 1,25-(OH)2D3 in LNCaP cells is associated with decreases in protein levels of Bcl-2 (29). Moreover, artificial overexpression of Bcl-2 abrogates 1,25-(OH)2D3-induced apoptosis, reduces the growth inhibitory capacity of 1,25-(OH)2D3, and allows for rapid recovery of LNCaP cells when hormone is removed (29). Thus, 1,25-(OH)2D3 action in inhibiting the growth of LNCaP cells appears to be mediated via both modulation of cell cycle and apoptotic processes. The relative contributions of cell cycle accumulation and the induction of apoptosis to the overall reduction in LNCaP cell number, compared with untreated controls, is not yet known. Because p53 plays an important role in both of these pathways, we wished to further examine the role of p53 in 1,25-(OH)2D3 action in prostate cancer cells. To assess any potential role for p53 in 1,25-(OH)2D3 induced growth inhibition, we studied 1,25-(OH)2D3 action in both PC-3 cells and an LNCaP-derived cell line, LN-56, in which p53 was rendered nonfunctional by expression of a dominant negative fragment of p53 (32, 33). Using these two cell lines, we found that although p53 is not required for the ability of 1,25-(OH)2D3 to extensively inhibit the growth of LNCaP prostate cancer cells mediated by apoptosis and G1 cell cycle accumulation, it is required for inducing LNCaP G0 arrest and for the synergistic effects of 1,25-(OH)2D3 and 9-cis retinoic acid.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
1,25-(OH)2D3 was obtained from Solvay Pharmaceuticals, Inc. DuPhar (Weesp, The Netherlands). The 9-cis retinoic acid was a gift from Drs. Elizabeth Allegretto and J. Wesley Pike (Ligand Pharmaceuticals, Inc., San Diego, CA). Phorbol 12-myristate 13-acetate (TPA) was obtained from Sigma (St. Louis, MO), and lovastatin was a gift of Dr. Marco Marcelli (Baylor College of Medicine, Houston, TX). Hormones were maintained as stock solutions in ethanol and stored in the dark at -20 C. Coulter Counter model ZF was from Coulter Cytometry (Hialeah, FL). Isoton and Zapoglobin II were from Coulter Corp. (Miami, FL), and the Profile I flow cytometer was from Coulter Electronics (Hialeah, FL). Hanks’ balanced salt solution without calcium or magnesium, PBS without calcium and magnesium and trypsin-EDTA (0.05% and 0.25%) were purchased from Life Technologies, Inc. (Rockville, MD). Nitrocellulose was from Schleicher \|[amp ]\| Schuell, Inc. (Keene, NH). Rabbit antimouse IgG was obtained from Zymed Laboratories, Inc. Corp. (South San Francisco, CA), and both horseradish peroxidase-conjugated antirabbit IgG and enhanced chemiluminescence (ECL) reagents were from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ). Tissue culture supplies were obtained from Fisher Scientific (Pittsburgh, PA). All chemicals were reagent grade unless otherwise indicated.

Cell culture
LNCaP cells were from American Type Culture Collection (Manassas, VA) and were maintained in RPMI containing 10% fetal calf serum (FCS) (Intergen Co., Purchase, NY) with penicillin and streptomycin (Life Technologies, Inc.). LN-56 cells were derived from LNCaP cells that were stably transfected with the genetic suppressor element 56 (GSE 56) as described by Rokhlin et al. (33). LN-56 cells were grown in RPMI containing 10% FCS, 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, and 400 µg/ml G418. PC-3 cells were also obtained from ATCC and were maintained in DMEM/F12 with 10% FCS and penicillin/streptomycin. All cell lines were maintained at 37 C in a humid atmosphere containing 5% CO2. Cells were trypsinized and plated 1 d before the start of each experiment.

Cell growth assay
Cells were seeded at low density and allowed to grow for the periods of time indicated in the figure legends. Hormones were added such that the concentration of ethanol did not exceed 0.1%, and control cells were treated with ethanol alone (vehicle) at the same concentration. Media and hormone treatments were replaced every 3 d. Using a Coulter Counter (Coulter Cytometry), total number of cells per well was determined as described previously (28). Each data point represents the mean of triplicate samples and one-way ANOVA as computed with the Sigma Stat program (Jandel Scientific, San Rafael, CA) was used to determine statistical significance.

Ki67 immunofluorescence
Cells were plated on coverslips (15,000 per well for PC-3 cells and 25,000 per well for LNCaP and LN-56 cells) and were treated for 6 d (with media change on d 3) with either 100 nM 1,25-(OH)2D3 or ethanol. Cells were then fixed in 100% ethanol at -20 C for 5 min and stored for up to 1 wk in 70% ethanol at 4 C. Cells were then stained with a mouse monoclonal antibody to Ki67 (Immunotech, Marseille, France) as described previously (29). Each experiment was performed at least three times, and a representative experiment is shown.

Analysis of retinoid receptor levels in PC-3 cells
Levels of retinoid X receptors (RXRs) and retinoic acid receptors (RARs) in PC-3 cells were determined using isotype-specific antibodies to immunoprecipitate receptors bound to [3H]-labeled 9-cis retinoic acid as described previously (28).

Cell cycle analysis
LN-56 or LNCaP cells were plated at 175,000 cells per 10-cm dish and allowed to attach overnight. Cells were treated with ethanol or 1,25-(OH)2D3 (10–100 nM) for 6 d. On the sixth day, cells were pulse labeled with 10 µM 5-bromo-2'-deoxyuridine for 6 h (LN-56) or 8 h (LNCaP) and then harvested for cell cycle analysis as described previously (28). Samples were analyzed using a Profile I flow cytometer. The experiment was performed at least three times, and a representative experiment is shown.

Cell death ELISA
LNCaP were plated at an initial density of 200,000, LN-56 cells at 175,000, and PC-3 cells at 125,000 cells per 10-cm dish. Cells were treated for 7 d with vehicle or 100 nM 1,25-(OH)2D3 with a change of medium on d 3. Cells were then scraped into their medium, pelleted, and washed with RPMI plus 10% FCS. Apoptosis was measured in equal number of cell equivalents, as directed in the manufacturer’s instructions using a cell death detection ELISA (Roche Molecular Biochemicals, Indianapolis, IN) to quantify cytoplasmic histone-associated DNA fragments in LNCaP, LN-56, and PC-3 cells. Treatments of LNCaP and LN-56 cells with 10 nM TPA (48 h) or PC-3 cells with 10 µM lovastatin (4 d) served as positive controls in the assay. To determine whether programed cell death was caspase dependent, LNCaP and LN-56 cells were plated at 175,000 cells per 10-cm plate and were pretreated with 10 µM zVADfmk or vehicle dimethylsulfoxide for 4 h before the addition of 1,25-(OH)2D3 or ethanol for 6 d. The experiments were repeated a minimum of three times with a representative example shown.

Western blot analysis

Bcl-2
LNCaP and LN-56 cells were initially plated at a density of 150,000 and 125,000 cells/10-cm dish, respectively. Cells were then treated for 6 d with 100 nM 1,25-(OH)2D3 or ethanol and harvested by scraping into the media. Following one wash in PBS, cells were lysed in a buffer containing 1% Triton X-100, 150 mM NaCl, 25 mM Tris (pH 7.4), with 1 µg/ml protease inhibitors (leupeptin, antipain, aprotinin, benzamidine HCl, chymostatin, and pepstatin) and 2 mM phenylmethylsulfonyl fluoride. After a 1-h incubation at 4 C, lysates were spun for 10 min at 12,000 rpm in a microcentrifuge. A Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA) was used to determine total protein concentration. Equivalent amounts of lysates were separated by 12.5% SDS-PAGE and transferred to nitrocellulose by overnight liquid transfer. Bcl-2 was detected with a mouse monoclonal antibody from DAKO Corp. (Carpinteria, CA) according to manufacturer protocol. Western blots were processed as previously described (29) and developed using ECL. Blots were simultaneously probed with a monoclonal antiactin antibody (Roche Molecular Biochemicals) as a loading control.

p53, p21, and c-Myc
Cells were seeded as noted in the individual figure legends and treated for the specified time points with ethanol or 100 nM 1,25-(OH)2D3. Cells were pelleted, washed in PBS without calcium or magnesium, and lysed by repeated freezing/thawing in 1x 10 mM Tris, 1 mM EDTA, 12 mM monothioglycerol (pH 7.4) with 0.4 M sodium chloride and protease inhibitors (1 µg/ml leupeptin, antipain, aprotinin, benzamidine HCl, chymostatin, pepstatin, and 2 mM phenylmethylsulfonyl fluoride). Lysates were collected by centrifugation at 12,000 rpm for 10 min at 4 C in a microcentrifuge. Total protein was determined using a Bradford protein assay and equivalent amounts of total protein were loaded on SDS-PAGE and transferred to nitrocellulose by overnight liquid transfer. Blots were blocked in 0.2% Tween-20 (TBST) containing 1% nonfat dried milk (TBST-milk) and incubated with a monoclonal anti-p53 antibody (Neomarkers, Union City, CA), a monoclonal anti-p21 antibody (Calbiochem-Novabiochem, San Diego, CA) or a monoclonal anti-c-Myc antibody (Roche) overnight at 4 C in TBST-milk. Blots were then washed and incubated with 0.2 µg/ml rabbit antimouse IgG secondary antibody in TBST-milk for 1 h at room temperature followed by washing and a final incubation with horseradish peroxidase-conjugated antirabbit IgG for 1 h at room temperature in TBST. The p21, p53, or c-Myc was then detected using ECL. Blots were simultaneously probed with a monoclonal antiactin antibody (Roche) to assure equal loading.

Transient transfection analysis
LNCaP and LN-56 cells were plated at 60% confluency in 10-cm dishes. Cells were then cotransfected in RPMI containing 10% FCS with 1 µg mdm2luc, a p53 reporter plasmid constructed in the laboratory of Dr. Guillermina Lozano (University of Texas M.D. Anderson Cancer Center, Houston, TX) and 150 ng cytomegalovirus-ß-galactosidase (as a control for transfection efficiency) using Fugene 6 transfection reagent (Roche) according to the manufacturer’s protocol. Twenty-four hours later, medium was replaced with fresh medium. Cells were treated with 100 nM 1,25-(OH)2D3 or vehicle (ethanol) for the 16 h before harvesting (48 h post transfection). Cells were lysed by incubation in 1 x reporter buffer (Promega Corp., Madison, WI) for 1 h at room temperature. Luciferase activity in 40 µl lysate was then measured using a Monolight 2010 model luminometer and luciferin substrate (Promega Corp.). Luciferase activity was normalized to ß-galactosidase expression in the same volume of lysate as a control for transfection efficiency.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LN-56 cells lack functional p53 protein
To address the role of p53 in the 1,25-(OH)2D3 response of LNCaP cells, we used LN-56 cells, a cell line derived from parental LNCaP cells using a vector expressing a dominant negative p53 fragment that disrupts p53 tetramerization and, consequently, p53 function (32, 33, 34). The dominant negative peptide expressed in LN-56 cells causes accumulation of nonfunctional p53 in rat embryo fibroblast cells (32) and LNCaP cells (33). As expected (Fig. 1AGo), expression of the dominant negative p53 peptide significantly increased p53 protein levels in the LN-56 cells, compared with the parental LNCaP cells.



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  		Figure 1. p53 is inactivated in LN-56 cells. A, Levels of p53 protein were measured by Western blot analysis. LNCaP and LN-56 cells were plated at 80% confluency and treated with either ethanol or 100 nM 1,25-(OH)2D3 for 72 h. Then 100 µg protein was separated on a 10% gel and subjected to Western blot analysis as described in Materials and Methods. B, LNCaP and LN-56 cells were transiently transfected with both a p53 reporter construct (mdm2luc) and cytomegalovirus-ß-galactosidase. Cells were treated for the final 16 h before harvesting with either ethanol (white bars) or 100 nM 1,25-(OH)2D3 (black bars). Reporter activity was determined by luciferase assay, and values have been normalized to transfection efficiency as described in Materials and Methods. E, Ethanol; D3, 1,25-(OH)2D3.

 
To confirm that p53 function was attenuated in LN-56 cells, the p53-dependent promoter of the mdm2 gene was used in a transient transactivation assay to assess the ability of endogenous p53 to transactivate this promoter. As shown in Fig. 1BGo, LNCaP cells transfected with the mdm2luc reporter plasmid express functional p53 as indicated by the presence of luciferase activity; 1,25-(OH)2D3 treatment does not alter p53’s ability to transactivate this promoter. In contrast, there was very little luciferase expression from the mdm2luc construct in LN-56 cells, confirming the absence of functional p53 in these cells. Taken together, the Western analysis for p53 and the transactivation assay indicate that LN-56 cells are devoid of functional p53 protein. These cells were therefore used as a tool to examine the role of p53 in LNCaP cell response to 1,25-(OH)2D3.

LN-56 cells are strongly growth inhibited by 1,25-(OH)2D3 but recover more rapidly than LNCaP cells
Previous studies have shown that LNCaP prostate cancer cells are strongly inhibited by 1,25-(OH)2D3, whereas PC-3 cells were only modestly inhibited (4). To determine whether loss of p53 would cause LN-56 cells to respond like PC-3 cells, we compared the growth of LNCaP, PC-3, and the LNCaP-derived LN-56 cells, which lack functional p53 (Fig. 2Go). Note that the LNCaP cell line is extensively growth inhibited by 1,25-(OH)2D3 (10 and 100 nM) when compared with cells treated with ethanol for 9 or 12 d. The PC-3 cells are less responsive to the inhibitory effects of 1,25-(OH)2D3 in that although their growth is slowed, they continue to proliferate in the presence of hormone (compare 10 and 100 nM 1,25-(OH)2D3 treatment at d 9 with d 12). In contrast to the PC-3 cells, LN-56 cells are extensively growth inhibited by 1,25-(OH)2D3 and do not continue to grow in the presence of 1,25-(OH)2D3. Thus, the response of LN-56 cells to 1,25-(OH)2D3 more closely mirrors the LNCaP cell response rather than the response of the p53-deficient PC-3 cells.



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  		Figure 2. Prostate cancer cells lacking functional p53 recover from 1,25-(OH)2D3 treatment. Cells were plated in 6-well plates at the following densities: 25,000 cells/well for LNCaP cells, 8,000/well for LN-56 cells, and 5,000/well for PC-3 cells. Cells were then treated with ethanol or 1,25-(OH)2D3 (10 or 100 nM) for the indicated periods of time. Recovery of treated cells was assessed by treating the cells with 1,25-(OH)2D3 for the first 6 d and replacing 1,25-(OH)2D3 with medium containing ethanol for either 3 (d 9 recovery) or 6 d (d 12 recovery). Bars represent the mean ± SD of triplicate samples. A representative experiment is shown. E, Ethanol; D3, 1,25-(OH)2D3; Rec, recovery. *, P <= 0.05, compared with ethanol treatment; {gamma}, P <= 0.05, compared with time-matched equivalent dose of 1,25-(OH)2D3 treatment.

 
We have shown that LNCaP cells treated with 1,25-(OH)2D3 recover poorly from treatment and exhibit a long delay in regaining normal growth rates when 1,25-(OH)2D3 is removed from the medium (29). To determine whether p53 plays any role in this resistance to recovery, we measured the response of LN-56 cells and PC-3 cells to the removal of 1,25-(OH)2D3. As demonstrated in Fig. 2Go, when LNCaP cells are treated with either 10 or 100 nM 1,25-(OH)2D3 for 6 d and then 1,25-(OH)2D3 treatment is withdrawn and replaced with vehicle, the cells fail to significantly recover from their extensive growth inhibition. In the recovery group, LNCaP cell numbers are comparable with those of cells that are subjected to continuous 1,25-(OH)2D3 treatment in agreement with our previous data (29). In contrast, both LN-56 cells and PC-3 cells, when treated with equivalent doses of 1,25-(OH)2D3 and subjected to this recovery assay, resume growth when compared with cells that were maintained in the presence of 1,25-(OH)2D3 during the entire experiment (Fig. 2Go). In LN-56 cells after 6 d of recovery, there is a 4-fold (at the 10 nM concentration) or 2.8-fold (at the 100 nM concentration) difference in cell number, compared with cells subjected to continuous 1,25-(OH)2D3 treatment. In PC-3 cells, the fold recovery is somewhat lower (1.4-fold after 3 d and 2.3-fold after 6 d in the 100 nM-treated cells) in part because of the fact that treated PC-3 cells are not completely inhibited by continuous 1,25-(OH)2D3 treatment as are the LN-56 cells.

Cells lacking functional p53 continue to express Ki67
We have shown previously that 1,25-(OH)2D3 greatly reduces the number of LNCaP cells expressing the Ki67 proliferation antigen when compared with control ethanol-treated cells (29). Ki67 nuclear antigen is expressed at varying levels in all phases of the cell cycle but not in quiescent cells (35, 36). Thus, it appears that 1,25-(OH)2D3 treatment does not merely induce G1 cell cycle arrest of LNCaP cells, but it may force the cells to enter a G0 state or quiescence, reducing the ability of the cells to recover from treatment. To determine whether p53 plays a role in this response, we examined Ki67 expression in 1,25-(OH)2D3 and ethanol-treated PC-3 and LN-56 cells using immunofluorescence. Figure 3Go shows total cell numbers (as determined by propidium iodide staining) and cells expressing Ki67. Almost none of the LNCaP cells (<1%) subjected to 1,25-(OH)2D3 treatment expressed Ki67 proliferation antigen, compared with ethanol-treated cells, in agreement with our previous findings (29). In contrast, a significant proportion of 1,25-(OH)2D3-treated LN-56 (>35%) and PC-3 (>90%) cells retain Ki67 expression. This is consistent with the rapid recovery from 1,25-(OH)2D3’s growth inhibitory effects by these two cell lines (Fig. 2Go).



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  		Figure 3. Lack of functional p53 reduces the ability of 1,25-(OH)2D3 to induce quiescence as measured by Ki67 staining in prostate cancer cells. LNCaP, LN-56, and PC-3 cells were stained for expression of Ki67 nuclear antigen. Cells were plated in 6-well plates at the following densities: 25,000/well for both LNCaP cells and LN-56 cells and 15,000/well for PC-3 cells; cells were treated with either ethanol or 100 nM 1,25-(OH)2D3 for 6 d and harvested for immunofluorescence as described in Materials and Methods. The left side of the panel shows cells that stain for Ki67 antigen; the right side shows the total number of cells in the field of view as detected by propidium iodide staining. E, Ethanol; D3, 1,25-(OH)2D3.

 
A role for p53 in synergism between 9-cis retinoic acid and 1,25-(OH)2D3 in growth-inhibiting prostate cancer cells
VDR forms heterodimers with other members of the steroid/thyroid nuclear receptor family, the RXRs, whose ligand is 9-cis retinoic acid (37). We showed previously that 9-cis retinoic acid acts synergistically with 1,25-(OH)2D3 to inhibit the growth of LNCaP cells (28). Because the PC-3 cells are much less responsive than the LNCaP cell line, we wanted to test whether we could increase the responsiveness of these cells by using combinations of 1,25-(OH)2D3 and 9-cis retinoic acid. As shown in Fig. 4Go, we treated PC-3 cells with combinations of 10 nM 1,25-(OH)2D3 and 1 nM or 10 nM 9-cis retinoic acid. We chose to use 10 nM 1,25-(OH)2D3 because it does not induce maximal inhibition of PC-3 cell growth (because it is less growth inhibitory than treatment with 100 nM 1,25-(OH)2D3). Using this suboptimal dose of 1,25-(OH)2D3, we show that combinations of 10 nM 1,25-(OH)2D3 and 9-cis retinoic acid are no more effective than treatment of the cells with 1,25-(OH)2D3 alone. Additionally, use of higher doses of 9-cis retinoic acid (100 nM) still does not result in synergistic effects of the two hormones in the PC-3 cells (data not shown). As a comparison with our previous findings, note that, compared with 1,25-(OH)2D3 treatment alone (27% of ethanol control), combinations of 9-cis retinoic acid and 1,25-(OH)2D3 are more effective [17% of control and 66% of 1,25-(OH)2D3 treatment alone] at growth-inhibiting LNCaP cells, although low levels of 9-cis retinoic acid alone are not growth inhibitory but actually stimulate cell growth (Fig. 4Go) (28).



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  		Figure 4. 9-cis Retinoic acid and 1,25-(OH)2D3 do not synergize in LN-56 or PC-3 cells. LNCaP (13,000 per well), PC-3 (3,000 per well), and LN-56 cells (8,000 per well) were treated with the indicated hormones for 9 d (LNCaP and PC-3) or 6 d (LN-56) and harvested as described in Materials and Methods. Each point represents the mean ± SD of triplicate samples. The inset in the LNCaP panel shows 10 nM 1,25-(OH)2D3 (left) and 10 nM 1,25-(OH)2D3 with 10 nM 9-cis retinoic acid (right). The experiment was performed at least three times, and a representative experiment is shown. E, Ethanol, D3, 1,25-(OH)2D3; 9C, 9-cis retinoic acid. *, P <= 0.05, compared with ethanol treatment; {gamma}, P <= 0.05, compared with 10 nM 1,25-(OH)2D3.

 
LNCaP cells express the full complement of RXRs as well as RARs (28). To determine whether the failure of PC-3 cells to respond to combinations of 9-cis retinoic acid and 1,25-(OH)2D3 was due to a lack of expression of RXRs, the levels of RXRs and RARs were measured in PC-3 cells (Table 1Go). The levels of RXRs and RARs were found to be similar to those previously reported for LNCaP cells (9–34 fmol per milligram of protein) (28). Therefore, it appears that the presence of both VDR and RXR is insufficient for 1,25-(OH)2D3 and 9-cis retinoic acid to synergistically inhibit the growth of prostate cancer cells.


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  		Table 1. Levels of retinoic acid and retinoid receptors in PC-3 cells

 
To determine whether the lack of functional p53 would affect synergism between 1,25-(OH)2D3 and 9-cis retinoic acid in LNCaP cells, we examined whether the LN-56 response to 1,25-(OH)2D3/9-cis retinoic acid combinations more closely resembles that of the LNCaP cells or PC-3 cells. Because the LN-56 cells are extensively growth inhibited by 9 d, we examined their response after 6 d of treatment (Fig. 4Go). The 9-cis retinoic acid alone does not inhibit growth and combinations of 10 nM 1,25-(OH)2D3, and either 1 or 10 nM 9-cis retinoic acid are no more effective than 10 nM 1,25-(OH)2D3 alone. Even combinations of 1,25-(OH)2D3 with 100 nM 9-cis retinoic acid do not show synergism in these cells (data not shown). However, treatment with 100 nM 1,25-(OH)2D3 inhibited growth more than 10 nM 1,25-(OH)2D3 treatment, showing that there is potential for additional growth inhibition of the cells. Longer treatment of cells (9 d) with these same hormone concentrations also does not show synergism (data not shown). Thus, the LN-56 cell response is similar to what we observed in the PC-3 cell line. Together, these data obtained in both the PC-3 and LN-56 cell lines suggest that p53 plays a role in the synergistic action between 1,25-(OH)2D3 and 9-cis retinoic acid in prostate cancer cells.

p53 Is not required for 1,25-(OH)2D3-induced G1 accumulation of LNCaP cells
1,25-(OH)2D3 induces a dose-dependent accumulation of the LNCaP cell line in the G0/G1 phase of the cell cycle (27, 28, 29). However, Campbell et al. (38) reported that PC-3 cells, although growth inhibited by 1,25-(OH)2D3, do not accumulate in the G1 phase of the cell cycle when treated with 1,25-(OH)2D3, and we have similar findings in PC-3 cells (data not shown). Because p53 plays an important role in cell cycle checkpoints, we measured 1,25-(OH)2D3’s effect on the cell cycle distribution of the LN-56 cell line. As shown in Table 2Go, treatment of LN-56 cells with 1,25-(OH)2D3 resulted in a dose-dependent G1 cell cycle accumulation, compared with ethanol-treated cells. Although this accumulation is not as dramatic as in the LNCaP cells in which 1,25-(OH)2D3 treatment resulted in 89% of the cells in G1 (Table 2Go), it is evident that p53 is not absolutely required for 1,25-(OH)2D3-induced G1 accumulation. Note that this assay does not distinguish between cells in the G0 and G1 phases of the cell cycle. Based on Ki67 data (Fig. 3Go) and the lack of cell recovery (Fig. 2Go), we believe that most of the LNCaP cells are in the G0 phase rather than in G1.


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  		Table 2. 1,25-(OH)2D3 increases the percentage of LN-56 cells in the G0/G1 phase of the cell cycle

 
p21 Protein levels are low, but regulated, in prostate cancer cells lacking p53
One of the major targets of p53 is the cyclin-dependent kinase inhibitor p21 (39, 40). Previous studies in LNCaP cells showed that p21 protein levels are modestly increased by 1,25-(OH)2D3 (27). Up-regulation of p21 has been implicated in 1,25-(OH)2D3’s induction of differentiation in leukemic cells (41, 42) and may play a role in the G1 accumulation induced by 1,25-(OH)2D3 in breast and prostate cancer cells (27, 43). To determine whether inactivation of p53 in LNCaP cells alters the ability of 1,25-(OH)2D3 to regulate p21 expression, we determined p21 protein levels in the LN-56 cells using Western blot analysis. As shown in Fig. 5Go, p21 protein is expressed in LN-56 cells and treatment of LN-56 cells with 1,25-(OH)2D3 increased its levels. 1,25-(OH)2D3 also increased p21 protein levels in PC-3 cells, in agreement with previous findings (38). Although others have reported regulation of p21 protein levels by 1,25-(OH)2D3 in the LNCaP cells (27, 44), the fold increase (2-fold) in expression is very small. We have found the induction of p21 protein and mRNA levels in LNCaP cells by 1,25-(OH)2D3 to be inconsistent (data not shown), which may be due to the already high basal levels of p21 expression in LNCaP cells. It is important to note that despite the higher levels of p21 expression in 1,25-(OH)2D3-treated LN-56 and PC-3 cells, the induced levels of p21 are considerably lower than basal expression levels in LNCaP cells (Fig. 5Go). Thus, although 1,25-(OH)2D3 regulates p21 protein levels in the absence of functional p53, the absolute levels of p21 are extremely low, compared with basal expression levels in the presence of p53.



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  		Figure 5. The p21 levels are low but regulated by 1,25-(OH)2D3, in prostate cancer cells lacking functional p53. LN-56 or PC-3 cells were plated in 10-cm dishes at 500,000 or 350,000 cells per dish, respectively, and were treated for the indicated times with either ethanol or 100 nM 1,25-(OH)2D3. Proteins (50 µg for the PC-3 blot and 100 µg for the LN-56 blot) were separated on 12.5% gels and subjected to Western analysis as described in Materials and Methods. As a comparison for expression levels, equivalent amounts of total protein (50 µg for the PC-3 blot and 100 µg for the LN-56 blot) from ethanol-treated LNCaP extracts were loaded on the same gel. Blots were probed with a monoclonal antibody to p21. Exposures were chosen to permit detection of p21 in LN-56 or PC-3 cells and are not for the same amount of time. E, Ethanol; D3, 1,25-(OH)2D3.

 
Interestingly, 1,25-(OH)2D3 induces G1 accumulation of LN-56 cells despite the fact that protein levels of p21 are very low in these cells. Thus, high levels of p21 are not required for G1 accumulation by 1,25-(OH)2D3 in prostate cancer cells. Cell cycle progression requires expression of numerous proteins. One protein required for growth and survival of many cell lines, including the LNCaP cells (45), is c-Myc. Because of reports of c-Myc regulation by 1,25-(OH)2D3 in some cell types (46, 47, 48), we asked whether 1,25-(OH)2D3 regulates c-Myc expression in prostate cancer cells. As demonstrated in Fig. 6Go, protein levels of c-Myc are decreased in LNCaP cells treated with 1,25-(OH)2D3 as early as 24 h after treatment. When the signal is normalized to actin levels, it is about 40% of control levels after 72 h of treatment. Levels of c-Myc are strongly decreased in the LN-56 cells after 72 h with 1,25-(OH)2D3 (reduced to 32% of control) and are also lower in PC-3 cells after 48 h of 1,25-(OH)2D3 treatment (reduced to 40% of control). In this experiment, c-Myc levels normalized to actin were not lower than control in PC-3 cells at 72 h, although we have seen some reduction in other experiments (data not shown). Thus, inhibition of prostate cancer cell growth by 1,25-(OH)2D3 is accompanied by a decrease in levels of c-Myc protein.



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  		Figure 6. 1,25-(OH)2D3 reduces levels of c-Myc protein in LNCaP, LN-56, and PC-3 cells. LNCaP (400,000 cells per 10-cm dish), LN-56 (500,000 cells per 10-cm dish), or PC-3 (350,00 cells per 10-cm dish) were treated with either ethanol or 100 nM 1,25-(OH)2D3 for the indicated periods of time. Fifty micrograms total protein from LNCaP cells or 75 µg from LN-56 or PC-3 cells were run on a 10% SDS-PAGE and subjected to Western blot analysis as described in Materials and Methods. Blots were then probed with a monoclonal antibody to c-Myc. E, Ethanol; D3, 1,25-(OH)2D3.

 
p53 Is not required for 1,25-(OH)2D3-induced apoptosis of LNCaP prostate cancer cells
We reported previously that 1,25-(OH)2D3 treatment induces apoptosis in LNCaP cells (29); to determine whether p53 plays a role in this response, we examined LN-56 and PC-3 cells for induction of apoptosis. LNCaP, LN-56, and PC-3 cells were treated with vehicle or 1,25-(OH)2D3 for 7 d, and fragmented DNA was detected with a cell death detection ELISA (Fig. 7AGo). Using this assay, we found that 1,25-(OH)2D3 induces apoptosis of LNCaP cells as measured by histone-associated DNA fragments, consistent with our previous results (29). Interestingly, in the absence of treatment, LN-56 cells have an increased basal rate of apoptosis when compared with the LNCaP cells. However, 1,25-(OH)2D3 treatment induces apoptosis in the LN-56 cells, although the fold increase is somewhat lower than in the LNCaP cells (2-fold, compared with 2.5-fold). In contrast, in the PC-3 cells, 1,25-(OH)2D3 does not induce cell death but seems to somewhat protect the cells from apoptosis. Use of TPA (LNCaP, LN-56) or lovastatin (PC-3) served as a positive control for induction of cell death (49, 50). To determine whether apoptosis induced by 1,25-(OH)2D3 is dependent on the activation of the caspase proteolytic cascade, we used the pan-caspase inhibitor zVADfmk in the cell death detection ELISA (Fig. 7BGo). 1,25-(OH)2D3 treatment caused a 4-fold induction in histone associated DNA fragments in the LNCaP cells, and this was completely inhibited by zVADfmk. In contrast, 1,25-(OH)2D3 caused a less than 2-fold increase in apoptosis of LN-56 cells; this appears to be partially blocked by zVADfmk, but the decrease is not statistically significant. However, TPA-induced apoptosis in the LN-56 cells was inhibited by zVADfmk as expected.



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  		Figure 7. 1,25-(OH)2D3 induces formation of histone-associated DNA fragments in LNCaP and LN-56 cells but not in PC-3 cells. A, LNCaP, LN-56, or PC-3 cells were treated for 7 d with either ethanol or 100 nM 1,25-(OH)2D3. Positive controls for the apoptosis assay included either 48 h of treatment with 10 nM TPA (LNCaP/LN-56) or 4 d of treatment with 10 µM lovastatin (PC-3). Histone-associated DNA fragments were detected using a cell death detection ELISA. *, P <= 0.05, compared with ethanol treatment (t test). B, LNCaP and LN-56 cells were pretreated with 10 µM zVADfmk or dimethylsulfoxide vehicle for 4 h before addition of 1,25-(OH)2D3 or ethanol vehicle for 6 d. Treatment with TPA for 48 h served as a positive control for the assay. Apoptosis was measured using an ELISA as in A. In both A and B, the left portion shows the fold induction relative to control, and the right side shows the absorbance measured in the ELISA. *, P <= 0.05, compared with ethanol treatment; {gamma}, P <= 0.05 treatment, compared with treatment with zVADfmk combination.

 
Bcl-2 protein levels are decreased by 1,25-(OH)2D3 in LN-56 cells
1,25-(OH)2D3 treatment decreases levels of the antiapoptotic protein Bcl-2 in LNCaP cells (29), and we have also shown that overexpression of Bcl-2 alters the growth properties of LNCaP cells and abrogates 1,25-(OH)2D3-induced apoptosis (29). Therefore, we determined whether levels of Bcl-2 were also regulated in the LN-56 cells. As shown in Fig. 8Go, levels of Bcl-2 were decreased after 6 d of treatment with 100 nM 1,25-(OH)2D3 in LN-56 cells. We have reported previously that 1,25-(OH)2D3 treatment reduces Bcl-2 levels in the PC-3 cells (29). Thus, in both p53-deficient LN-56 cells and PC-3 cells, Bcl-2 levels are down-regulated by 1,25-(OH)2D3. However, down-regulation of Bcl-2 is insufficient for 1,25-(OH)2D3 to induce apoptosis in the PC-3 cell line.



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  		Figure 8. 1,25-(OH)2D3 reduces Bcl-2 expression in both LNCaP and LN-56 cells. LNCaP or LN-56 cells were plated at 150,000 or 125,000 cells per 10-cm dish, respectively, and were treated for 6 d with ethanol or 100 nM 1,25-(OH)2D3. One hundred micrograms cellular lysate were subjected to 12.5% SDS-PAGE and probed with an antibody to Bcl-2 as described in Materials and Methods. E, Ethanol; D3, 1,25-(OH)2D3.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although numerous studies have shown that 1,25-(OH)2D3 inhibits the growth of many different types of cancer cells, including breast (1, 2), colon (3), and prostate cancer cells (4, 6, 7), the extent of growth inhibition varies widely even within a particular cancer cell type. The mechanisms by which 1,25-(OH)2D3 inhibit the growth of cancer cells are, for the most part, unknown. Up-regulation of the cyclin-dependent kinase inhibitor, p21, has been implicated in the differentiation of U937 myelomonocytic cells (41, 42). In contrast, induction of apoptosis has been implicated in 1,25-(OH)2D3-dependent inhibition of breast cancer cells (51, 52, 53) in a p53-independent manner (54). We and others have shown that 1,25-(OH)2D3 induces G1 accumulation of LNCaP cells (27, 28) and Zhuang and Burnstein (27) reported a concomitant increase in p21 protein. Additionally, our group found that 1,25-(OH)2D3 induces a 5-fold increase (to about 10%) in the percentage of LNCaP cells undergoing apoptosis with an accompanying decrease in Bcl-2 protein levels (29).

LNCaP cells are the least aggressive of the well-characterized prostate cancer cell lines, requiring androgens for their growth as well as growth factors supplied by Matrigel or stromal cells for growth in vivo (55, 56, 57, 58). We found, as reported by others (4), that 1,25-(OH)2D3 is less effective in inhibiting the growth of the androgen-independent PC-3 cells, compared with the LNCaP cells (Fig. 2Go). Zhuang et al. (11) have shown that additional expression of the vitamin D receptor in the PC-3 cells is not sufficient to improve the responsiveness of these cells. Although 1,25-(OH)2D3 induces 24-hydroxylase (the enzyme that converts 1,25-(OH)2D3 into an inactive metabolite) in PC-3 cells and not in LNCaP cells (4, 7), this difference alone does not explain the reduced response. We have found that more frequent addition of 1,25-(OH)2D3 or use of an analog (EB1089) that is not 24-hydroxylated in vivo (59) does not substantially alter the responsiveness of PC-3 cells (data not shown).

Among the many differences between LNCaP cells and the more aggressive androgen-independent PC-3 cells is the lack of functional p53 in PC-3 cells. To assess more directly the role of p53 in 1,25-(OH)2D3 action, we used an LNCaP-derived line of cells in which p53 function was eliminated. LN-56 cells do not have functional p53, as demonstrated by lack of activation of a p53-dependent promoter construct, mdm2luc, as well as by accumulation of p53 protein, a characteristic of expression of this particular dominant negative peptide in cells (32, 33). Furthermore, LN-56 cells are resistant to death induced by Adriamycin, a drug that requires functional p53 to kill cells (our unpublished results). LN-56 cells are therefore a better-defined model than the PC-3 cells for dissecting the role of p53 in 1,25-(OH)2D3 action in prostate cancer cells.

As shown in Fig. 2Go, we found that 1,25-(OH)2D3 inhibits the growth of LN-56 cells nearly as extensively as the LNCaP cells, suggesting that 1,25-(OH)2D3 does not require p53 to exert potent growth inhibitory effects in prostate cancer cells. However, of interest is our observation that combinations of 9-cis retinoic acid and 1,25-(OH)2D3 do not inhibit growth more effectively than 1,25-(OH)2D3 in the LN-56 and PC-3 cells as they do in the LNCaP cells. This lack of synergism between 1,25-(OH)2D3 and 9-cis retinoic acid is not due to lack of RXR expression in PC-3 cells, which express the full complement of RXRs (Table 1Go). Taken together, these data suggest that p53 plays some role in the functional synergism that is observed between 1,25-(OH)2D3 and 9-cis retinoic acid.

The growth inhibition of the LN-56 cells, like that of the LNCaP cells, involves an increase of cells in the G1 phase of the cell cycle (Table 2Go) that is accompanied by an increase in the protein levels of the p21 cyclin-dependent kinase inhibitor. However, the induced levels of p21 are very low, compared with the LNCaP cells (Fig. 5Go), most probably caused by the lack of p53 in LN-56 cells because p21 is a p53 target gene (39). Because the levels of p21 protein are so low in LN-56 cells, we postulated that 1,25-(OH)2D3 might be regulating some other gene(s) that plays a critical role in cell cycle progression. One potential gene is c-Myc. Levels of c-Myc are rapidly induced in quiescent cells when growth factors are introduced into their environment (60, 61, 62). Additionally, removal of c-Myc expression by antisense strategies or deletion of the gene causes a lengthening of the cell cycle (63, 64). c-Myc is regulated by 1,25-(OH)2D3 in some other cell types (46, 47, 48), and reduction of c-Myc proteins levels by antisense oligonucleotides in LNCaP cells is growth inhibitory (45). As shown in Fig. 6Go, c-Myc protein levels are decreased in all three prostate cancer cell lines, with the greatest down-regulation observed in the LNCaP and LN-56 cells. This down-regulation of c-Myc by 1,25-(OH)2D3, rather than up-regulation of p21 expression levels, may be a major contributor to the G1 accumulation observed in the LNCaP and LN-56 cells. This finding is made more interesting by studies showing that in some tumors, down-regulation of c-Myc, even transiently, results in sustained tumor regression and differentiation of osteogenic sarcoma cells (65).

Despite the inhibitory effects of 1,25-(OH)2D3 on the growth and cell cycle of LN-56 cells, these cells share with PC-3 cells the ability to recover rapidly from treatment. Therefore, although p53 is not required for growth inhibition by 1,25-(OH)2D3, lack of functional p53 affects the ability of 1,25-(OH)2D3 to inhibit nearly irreversibly the growth of prostate cancer cells, allowing the cells to recover rapidly on withdrawal of the hormone. Interestingly, the irreversible growth-inhibitory effects of 1,25-(OH)2D3 in LNCaP, but not LN-56 and PC-3 cells, are correlated with retention of Ki67 expression. This suggests that the long-lasting effect of 1,25-(OH)2D3 in the LNCaP cells results from the cells exiting the cell cycle rather than simply being halted in the G1 phase. In the absence of functional p53, 1,25-(OH)2D3 treatment does not result in G0 arrest, allowing these cells to quickly regain normal growth capabilities when 1,25-(OH)2D3 is no longer present. These results support findings from Itahana et al. (66), who found that p53 plays an important role in establishing and maintaining growth arrest in fibroblasts subjected to growth factor withdrawal.

As judged by the results of the cell death ELISA, p53 is not absolutely required for the induction of apoptosis in LNCaP-derived prostate cancer cells. However, the lack of p53 alters the responsiveness (Fig. 7Go). The fold increase in absorbance is consistently lower in the LN-56 cells. Interestingly, although we were able to detect apoptosis in LNCaP cells using terminal transferase and flow cytometry (29), our attempts to detect apoptosis in the LN-56 cells using this assay were consistently unsuccessful. The increase in signal over the high background levels of apoptosis in the LN-56 cells was not statistically significant (data not shown). Our finding is in agreement with results from studies of 1,25-(OH)2D3 action in breast cancer cells in which p53 does not appear to be required for 1,25-(OH)2D3 to induce apoptosis (54). We also show here that the induction of programed cell death in the LNCaP is caspase dependent because a pan-caspase inhibitor blocks cell death induced by 1,25-(OH)2D3 in these cells. In contrast, apoptosis induced by 1,25-(OH)2D3 in the LN-56 cells may be only partially caspase dependent because zVAD-fmk does not fully block cell death in LN-56 cells. Studies in breast cancer cells have demonstrated that 1,25-(OH)2D3 can induce caspase-independent cell death via calcium and calpain (67), and this may also be true in prostate cancer cells. Our studies also show that despite the lack of p53, a known negative regulator of the bcl-2 gene, Bcl-2 protein levels are decreased in the LN-56 cells after treatment with 1,25-(OH)2D3. In contrast to our results in the LNCaP and LN-56 cells, 1,25-(OH)2D3 does not induce apoptosis in the PC-3 cells. However, this difference may not simply be due to p53 inactivation in the PC-3 cells because these cells likely lack proteins other than p53, which result in their differing response to 1,25-(OH)2D3.

Collectively, these studies show that p53 is not required to induce extensive growth inhibition by 1,25-(OH)2D3, to cause accumulation in the G1 phase of the cell cycle or induce some level of apoptosis in LNCaP cells. Because induction of apoptosis by 1,25-(OH)2D3 in the LNCaP and LN-56 cells is relatively low, it is likely that the most important mechanism of growth inhibition of the cells by 1,25-(OH)2D3 is through its ability to cause these cells to accumulate in the G1 phase of the cell cycle. However, elimination of p53 function in the LNCaP cells reduces G0 arrest as measured by loss of Ki67 expression, allows the cells to recover from 1,25-(OH)2D3 treatment, and eliminates the growth-inhibitory effects of combinations of 9-cis retinoic acid and 1,25-(OH)2D3. Thus, additional as-yet-undetermined differences, not simply lack of p53, cause the PC-3 cells to be much less growth inhibited than either the LNCaP or LN-56 cells.


   Acknowledgments
 
The authors acknowledge Andrei Gudkov for making the LN-56 cell line, Oskar Rokhlin for assistance, Dorothy Lewis and the Flow Cytometry Core at Baylor College of Medicine for helpful suggestions with cell cycle and apoptosis experiments, and Judy Roscoe in the Tissue Culture Core for help with cell culture.


   Footnotes
 
This work was supported by NIH Grant CA-75337 (to N.L.W.); National Cancer Institute Specialized Programs of Research Excellence Grants for Prostate Cancer CA-58204 (to N.L.W.) and CA-76673 (to M.B.C.); National Research Service Award F3283277 (to L.V.S.); and Molecular Endocrinology Training Grant T32-DK-07696 (to T.C.P.).

Abbreviations: ECL, Enhanced chemiluminescence; FCS, fetal calf serum; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; RAR, retinoic acid receptor; RXR, retinoid X receptor; TBST, 0.2% Tween-20; TBST-milk, TBST containing 1% nonfat dried milk; TPA, phorbol 12-myristate 13-acetate; VDR, vitamin D receptor.

Received February 1, 2001.

Accepted for publication September 23, 2002.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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