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Endocrinology |
, 25-Dihydroxyvitamin D3 Are Mediated by Increased Levels of p21 in the Prostatic Carcinoma Cell Line ALVA-311
University of Colorado Health Sciences Center, Department of Pathology, Denver, Colorado 80262
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 25-Dihydroxyvitamin D3 [1, 25-(OH)2D3] is recognized to have significant antiproliferative effects on certain prostatic carcinoma (PC) cell lines, although the precise mechanisms of action remain in question. We have evaluated the role of the cell cycle-dependent kinase inhibitor p21. In the PC cell lines ALVA-31 and LNCaP, 1, 25-(OH)2D3 inhibits growth and induces both p21 mRNA and protein levels. Growth inhibition of ALVA-31 cells was abolished by stable transfection with a p21 antisense construct. This effect was not attributable to a reduction in functional vitamin D receptors as measured by transcriptional activity with a luciferase-vitamin D response element reporter construct. Therefore, increased p21 expression appears necessary to mediate the antiproliferative effects of this hormone in ALVA-31 cells. Cell lines that are insensitive to the growth inhibitory properties of 1, 25-(OH)2D3 failed to up-regulate p21 expression after hormone treatment; these include sublines of ALVA-31 as well as the cell lines TSU-Pr1 and JCA-1. In the latter two lines, adenovirus-mediated expression of a sense p21 cDNA significantly reduced their proliferation as compared with a control adenoviral construct. This suggests that the signaling pathway downstream of p21 is intact in TSU-Pr1 and JCA-1 cells, although p21 expression appears unregulated by 1, 25-(OH)2D3. We propose a model in which the antiproliferative effect of 1, 25-(OH)2D3 on PC cells is mediated through increased p21 expression. Elucidation of why this effect is absent in select cell lines may provide valuable insight into the variability of responses observed in PC patients treated with vitamin D. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The classical actions of 1, 25-(OH)2D3, the hormonally active form of vitamin D3, are to regulate calcium and phosphate homeostasis and thereby promote the mineralization of bone (4)
. However, in recent years it has become increasingly apparent that 1, 25-(OH)2D3 is also able to elicit physiological responses in extraosseous cell types, leading to a wide array of actions. These nonclassical actions include the regulation of cellular proliferation, differentiation, and immunosuppression (5, 6, 7, 8)
. The majority of the actions of 1, 25-(OH)23 are mediated through a nuclear VDR, a member of the steroid/thyroid superfamily of ligand-activated transcription factors (9)
. On binding of 1, 25-(OH)2D3 to the VDR, the activated complex commonly heterodimerizes with the retinoid X receptor. This dimer is capable of binding to VDREs in the promoter regions of target genes leading to activation or repression of transcription via interaction with transcriptional cofactors and the basal transcriptional machinery (10)
.
There is now considerable evidence suggesting a role for 1, 25-(OH)2D3 in protecting against human cancer. Many different malignant tissues and cell types express nuclear VDRs and are sensitive to the antiproliferative effects of 1, 25-(OH)2D3 both in vitro and in vivo (11, 12, 13)
. Reduced serum levels of 1, 25-(OH)2D3 in humans have been associated with an increased risk of breast (14)
, colon (15)
, and prostate (16)
cancer. Increased serum levels of 1, 25-(OH)2D3-binding protein (17)
and polymorphisms in the VDR gene (18
, 19)
have also been associated with an increased risk of PC. Furthermore, clinical data indicate that patients with advanced-stage PC are often vitamin D3 deficient (20)
.
Although it is widely accepted that 1, 25-(OH)2D3 decreases the growth of several malignant cell types, the precise mechanism(s) through which it acts remains unclear. It has been shown that 1, 25-(OH)2D3 can induce apoptosis of breast (13)
, colon (21)
, and leukemic (22)
cell lines. However, this remains controversial and does not appear to be the case in the majority of PC cell lines4
(23, 24, 25)
. An isolated report describes vitamin D-mediated growth inhibition occurring via the androgen receptor (26)
. However, several lines of evidence argue against this. First, 1, 25-(OH)2D3 has an antiproliferative effect in two androgen receptor-negative PC cell lines (12)
. Secondly, in the cell line LNCaP, 1, 25-(OH)2D3 cannot compete with the binding of the synthetic androgen R1881, even at a 250-fold molar excess concentration (27)
. Lastly, based on transfection analyses, the expression of the nuclear VDR has been shown to be necessary and sufficient to mediate the antiproliferative effects of 1, 25-(OH)2D3 in two cell lines that do not express androgen receptors (28
, 29)
. These data indicate that whereas the nuclear VDRs are required for growth inhibition of PC cells by 1, 25-(OH)2D3, androgen receptors are not.
In the PC cell line LNCaP, 1, 25-(OH)2D3 has been shown to regulate the cell cycle at various levels (30
, 31)
. Specifically, 1, 25-(OH)2D3-mediated growth inhibition has been correlated with an increase of cells in the G0/G1 phase of the cell cycle, increased levels of the cell cycle-dependent kinase inhibitors p21 and p27, increased CDK2 activity, and retinoblastoma hypophosphorylation (23)
. The regulation of p21 is complex, involving both transcriptional and post-transcriptional mechanisms (32)
. In HL-60 leukemia cells, p21 is transcriptionally induced by 1, 25-(OH)2D3 in a VDR-dependent but not p53-dependent manner (33)
. A VDRE in the promoter region of p21 has been identified (34)
. However, direct activation of p21 by 1, 25-(OH)2D3 is not a uniform finding in all of the cell types (23)
. In most cases, a direct relationship between the changes in p21 expression and the vitamin D-induced antiproliferative response has not been shown.
In the current study we directly analyzed the role of p21 in mediating the antiproliferative effects of 1, 25-(OH)2D3 in PC cells. We found that neither the basal level of p21 message nor p21 protein correlated with 1, 25-(OH)2D3 growth responsiveness. Both the p21 message and protein levels were up-regulated after treatment with 1, 25(OH)2D3 in PC cell lines that are responsive to the growth inhibitory effects of the hormone. Likewise, in those cells where growth is not inhibited by 1, 25-(OH)2D3, no regulation of p21 was apparent. Stable transfection of antisense p21 cDNA into the 1, 25(OH)2D3-responsive ALVA-31 PC cell line resulted in abrogation of the growth inhibitory effect of the hormone. Furthermore, in two cell lines that are not sensitive to the antiproliferative effects of 1, 25(OH)2D3, p21 transgene expression by itself inhibits their growth, suggesting that the signaling pathway downstream of p21 remains intact in these cells. We conclude that up-regulation of p21 expression plays an active and necessary role in mediating growth inhibition by 1, 25-(OH)2D3 in PC cell lines.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vitamin D.
1, 25-(OH)2D3 was a generous gift from B. Borsje (Solvay Duphar B.V., Weesp, The Netherlands). Stock solutions in absolute ethanol were stored as concentrates at -20°C. Working dilutions of 1, 25-(OH)2D3 were freshly made in supplemented RPMI 1640 before each experiment. The ethanol concentrations in each test condition never exceeded a final concentration of 0.1% (v/v).
Growth Inhibition Assays.
The cell lines were transferred to multiwell plates at the following densities: LNCaP, 1 x 103/cm2; and ALVA-31, TSU-Pr1, and JCA-1, 0.25 x 103/cm2. The cells were plated in medium containing 10% FBS and were left to attach to the surface overnight. The plating medium was removed and replaced with medium containing either ethanol vehicle or 10-8 M 1, 25-(OH)2D3. The cells were then allowed to grow for an additional 4 days. Total DNA content of the cells in each well was determined using the Hoechst 33258 assay as described previously (43)
. The percentage inhibition of growth as compared with the ethanol vehicle control was calculated as the difference between the amount of DNA present in the treated cells versus control samples divided by the DNA content of the control. Each experiment was carried out in triplicate, and the experiments were repeated at least three times.
Flow Cytometric Analysis of Cell Cycle.
For the plating density experiments, LNCaP cells were plated in T-25 tissue culture flasks at 1,300, 2,500, 4,000, 8,000, and 12,000 cells/cm2 in RPMI 1640 with 10% FBS. At the indicated time points (24 and 72 h), the medium was replaced with 5 ml of fresh medium containing 10-8 M 1, 25-(OH)2D3 or 0.1% (v/v) ethanol vehicle. The medium was replaced every 48 h after the initial feeding. All of the samples were harvested simultaneously by pooling the floating cells with the trypsinized monolayers and pelleting the cells by centrifugation at 600 rpm (70 x g) for 5 min. Next, the cells were resuspended in a solution containing 25 µg/ml propidium iodide (Sigma Chemical Co., St. Louis, MO), 10 mM EDTA, 0.1 mg/ml DNase-free RNase, and 0.3% saponin in PBS (pH 7.4). The samples were incubated in this solution for 16–24 h at 4°C before performing cell cycle analysis using a Coulter XL flow cytometer (Coulter Corp., Miami, FL). Doublet correction was achieved by gating on peak fluorescence versus integral fluorescence. Statistics were performed on 10,000 events/sample using Modfit 5.2 software (Verity Software House, Inc., Topsham, ME).
For the experiments that varied the length of time in culture before hormone treatment, LNCaP cells were plated in T-25 flasks at 2500 cells/cm2 in RPMI 1640 with 10% FBS. After 24 h, flasks were divided into two groups for treatment. In group A, all of the flasks were treated at the same time with medium containing ethanol vehicle or 10-8 M 1, 25-(OH)2D3. Cells were then harvested at varying time points after treatment (24 and 72 h) for flow cytometric analysis of cell cycle distribution as described above. In group B, cells were treated with ethanol vehicle or 10-8 M 1, 25-(OH)2D3 at varying time points (24 and 72 h) and were harvested simultaneously. Each condition was performed in triplicate flasks, and the graphed values represent the mean of triplicate samples ± SE, where 5000 events were counted in each sample.
Real-time Quantitative RT-PCR.
Real-time quantitative RT-PCR (TaqMan PCR) was performed according to the manufacturer’s protocol using an ABI PRISM 7700 Sequence Detection System and a TaqMan PCR Core Reagent kit (Perkin-Elmer Corp., Foster City, CA). Total cellular RNA was isolated from cells at 
80% confluency using a guanidine isothiocyanate method (44)
. Reverse transcription was performed on 1 µg of total RNA treated with DNase I (Life Technologies, Inc.). The forward p21 primer was 5'-TGGAGACTCTCAGGGTCGAAA-3'. The reverse primer was 5'-CGGCGTTTGGAGTGGTAGAA-3'. A specific 6FAM-CGGCGGCAGACCAGCATGAC-TAMRA probe was used to quantify amplification. Conditions for quantitative real-time PCR were as follows: 48°C for 2 min; 95°C for 10 min followed by 40 cycles of 95°C for 15 s; and 60°C for 1 min. The amplification plots of p21 cDNA were used to discover the threshold cycle, which represents the PCR cycle where an increase in reporter fluorescence above baseline level is first detected. Comparison of the experimental results to a standard curve provided quantitation of the input RNA. The amount of p21 RNA was normalized to the amount of rRNA in each sample. SE for each data point was generated using data from at least three separate experiments.
Western Blot Analyses.
Cell cultures were used at 80% confluency. Total cell lysates were prepared in radioimmunoprecipitation assay buffer plus a Protease and Phosphatase Inhibitors mixture (Sigma Chemical Co.) and quantitated using the Bradford Assay (Bio-Rad, Melville, NY). Aliquots of total protein (100–200 µg) were subjected to electrophoretic analysis through a 15% SDS-PAGE gel. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (NEN). The completed transfer was evaluated using prestained protein standards (Bio-Rad). After blocking nonspecific binding with 5% powdered milk in 0.1% phosphate buffered saline with 0.1% Tween 20, the membrane was incubated with a mouse monoclonal anti-p21 primary antibody (Dako Corp., Carpenteria, CA) at a concentration of 0.78 µg/ml (a 1:500 dilution). Membranes were subsequently incubated with a 1:5000 dilution of sheep antimouse secondary antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech, Piscataway, NJ). Signal was detected using enhanced chemiluminescence (Amersham Pharmacia Biotech). Experiments were performed at least three separate times.
Recombinant p21 Constructs.
The pcDNA3-p21 plasmid was kindly provided by A. Kraft, University of Colorado Health Sciences Center, Denver, CO. It was digested using HindIII and XhoI (Promega, Madison, WI) to release a 2.1-kb fragment of the human p21 sequence that included the entire coding region and part of the 3' untranslated region. After treatment with Klenow polymerase (Boehringer Mannheim, Indianapolis, IN), the p21 fragment was inserted into the HpaI site of the LNCX retroviral vector (45)
using a modified ligation technique as described previously (28)
. The DNA was then redigested with HpaI to linearize any religated vector not containing the p21 sequence and was purified via phenol-chloroform extraction and ethanol precipitation.
Competent Escherichia coli (Invitrogen Corp., Carlsbad, CA) were transformed using the phenol-chloroform-purified DNA. Ampicillin-resistant colonies were screened using a colony lift protocol with an [-32P]dATP-labeled fragment of p21. Minipreps of the positive plaques were digested with HindIII and BglII (Promega) to determine sense versus antisense orientation of the p21 sequence. Selected bacterial colonies were propagated, and plasmid DNA was purified using a MaxiPrep kit (Promega).
Stable Transfections.
ALVA-31 cells at passage 25 were electroporated using the BRL Cell-Porator (Life Technologies, Inc.) with either the LNCX vector or the LNCX antisense p21 construct as described previously (28)
. The transfected cells were maintained in the presence of 0.25 mg/ml Geneticin (Life Technologies, Inc.) to discourage spontaneous reversion.
Transient Transfection and Reporter Gene Assays.
ALVA-31 parental cells, ALVA-31 cells transfected with empty LNCX vector, and ALVA-31 cells transfected with antisense p21 cDNA were transiently transfected with a VDRE-containing fragment of the 24-hydroxylase promoter attached to a firefly luciferase reporter plasmid (kindly provided by Dr. R. Heyman, Ligand Pharmaceuticals, San Diego, CA) and a ß-galactosidase reporter plasmid (46)
using Lipofectin (Life Technologies, Inc.) according to the manufacturer’s instructions. Lipofectin (10 µl) was used per 1 µg of transfected DNA. The Lipofectin and DNA were incubated separately for 45 min in RPMI 1640 at room temperature. The solutions were combined and allowed to stand for an additional 15 min. The normal growth medium was removed from the cells, replaced with medium containing the Lipofectin:DNA mixture, and the cells incubated for 16–24 h. The medium was replaced with RPMI 1640 plus 10% FBS containing an ethanol control or 10-8 M 1, 25-(OH)2D3. Cells were harvested for luciferase and ß-galactosidase activity after 48 h. Data are presented as the average fold activity of 1, 25-(OH)2D3 treated cells versus untreated controls.
Adenoviral Transduction of Sense p21.
Recombinant adenoviruses were kindly provided by Dr. J. Degregori (University of Colorado Health Sciences Center, Denver, CO). In these viruses, the E1A and E1B sequences have been replaced with either the green fluorescent protein cDNA (47)
or the p21 cDNA (48)
, each under the control of the cytomegalovirus promoter. JCA-1 and TSU-Pr1 cells were transduced using 50 pfu/cell of Ad-p21 or Ad-GFP, as described previously (47)
. The transduced cells were plated in triplicate in 24-well tissue culture dishes and were incubated at 37°C. The tissue culture medium was replaced every 48 h, and cells were harvested at the designated time points by aspirating the medium and lysing the monolayers in 0.5 M NaOH. The DNA contents of the monolayers were quantified by Hoechst 33258 fluorescence (43)
.
Statistical Analyses.
Comparisons between the growth inhibitory effects of drug treatment and controls and analyses of the differences between subgroups of different cell lines were performed using parametric one-way ANOVA. Differences between groups were interpreted as being statistically significant when Ps were <0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 25-(OH)2D3., 25-(OH)2D3 on the PC cell lines ALVA-31 and LNCaP (12
, 23 , 28
, 30
, 49)
. Whereas two reports suggest that the antiproliferative effect of 1, 25-(OH)2D3 on LNCaP cells can be attributed to an increase in the percentage of cells in the G0/G1 phase of the cell cycle (23
, 30)
, significant variation exists between these reports and between our preliminary findings. Two factors that could possibly effect the ability of 1, 25-(OH)2D3 to alter cell cycle distribution are initial plating density of the cells and the length of time cells are in culture before they are treated with of 1, 25-(OH)2D3. This prompted us to carefully analyze these parameters on the outcome of cell cycle distribution experiments with 1, 25-(OH)2D3.
LNCaP cells were plated at five different densities varying from 1,300 to 12,000 cells/cm2, which spanned the densities used in previous reports. After 24 or 72 h, cells were treated with medium containing ethanol vehicle or 10-8 M 1, 25-(OH)2D3. All of the samples were then harvested simultaneously for flow cytometric determination of cell cycle distribution. The results shown in Table 1
indicate that G0/G1 accumulation in response to 1, 25-(OH)2D3 can be detected earlier and is progressively greater in magnitude with increased plating density. Using these methods, the effects are most apparent 72 h after treatment.
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, 25-(OH)2D3. In one group, LNCaP cells were allowed to attach overnight, and then all of the samples were treated with medium containing either ethanol vehicle or 1, 25-(OH)2D3. Samples were then harvested after various time points (Fig. 1A)
. In group B, cells were treated at varying times, and then all of the samples were harvested simultaneously (Fig. 1B)
.
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, 25-(OH)2D3 as early as 24 h after treatment, whereas protocol A does not. After 72 h of treatment, both protocols provide similar results. Together, the data presented in Table 1
and Fig. 1
indicate that both plating density and the length of time cells have been in culture before hormone treatment effect the sensitivity of LNCaP cells to 1, 25-(OH)2D3 in cell cycle distribution experiments. These results explain, at least in part, the great variation observed between laboratories but confirm that 1, 25-(OH)2D3 is indeed causing a significant proportion of cells to accumulate in G0/G1 without any evidence of apoptotic induction.
Similar studies have analyzed the effects of 1, 25-(OH)2D3 on cell cycle distribution of ALVA-31 cells, which are also sensitive to the antiproliferative effects of 1, 25-(OH)2D3. As with LNCaP cells, 1, 25-(OH)2D3 did not induce apoptosis in ALVA-31 (23)
. Interestingly, changes in ALVA-31 cell cycle distribution were also undetectable (23)
. These findings are in agreement with our unpublished observations. This suggests that 1, 25-(OH)2D3 may slow the rate of cell progression through S phase and G2-M, which cannot be measured by standard flow cytometric cell cycle analysis.
Basal Levels of p21 Expression Do not Correlate with Growth Responsiveness to 1, 25-(OH)2D3.
Numerous hormones and growth factors are known to regulate p21 expression levels. These include the transforming growth factor ß isoforms, sex steroids, glucocorticoids, and 1, 25-(OH)2D3 (32)
. To determine whether any correlation exists between the basal level of p21 mRNA expressed in a cell and its sensitivity to the growth inhibitory effects of 1, 25-(OH)2D3, we used real-time quantitative RT-PCR to quantify the amount of p21 mRNA in eight PC4
cell lines. Fig. 2A
shows the basal amount of p21 mRNA expressed in each of the cell lines. No correlation is evident between basal p21 mRNA expression and 1, 25-(OH)2D3 growth sensitivity. Whereas growth of both ALVA-31 and LNCaP cells is inhibited by 1, 25-(OH)2D3 treatment, the LNCaP cell line expresses the highest level of p21 mRNA whereas ALVA-31 cells express the lowest. TSU-Pr1 and JCA-1 cells, which are insensitive to the antiproliferative effects of 1, 25(OH)2D3, express intermediate levels of p21 as compared with LNCaP and ALVA-31.
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. It is evident that protein levels are not perfectly predicted by mRNA levels in every cell line. However, there is still no correlation between basal p21 expression levels and sensitivity to the growth inhibitory properties of 1, 25-(OH)2D3.
1, 25-(OH)2D3 Induction of p21 Levels Correlates with 1, 25-(OH)2D3-mediated Growth Responsiveness.
It has been proposed that the function of p21 within a cell may be determined by its level of expression (50)
. The differences we observed in expression levels of p21 mRNA between the various cell lines may, therefore, be the result of its pleiotropic functions and the need to maintain cellular homeostasis. Consequently, p21 mediation of the antiproliferative effects of 1, 25-(OH)2D3 would require an increase in the amount of p21 protein above steady state levels. This possibility led us to determine whether or not a correlation exists between the induction of p21 expression by 1, 25-(OH)2D3 and growth sensitivity. Fig. 3
shows the levels of p21 mRNA after timed treatment with 10-8 M 1, 25-(OH)2D3. It is clear that the two 1, 25-(OH)2D3 sensitive lines, ALVA-31 and LNCaP, up-regulate p21 mRNA in response to 1, 25-(OH)2D3 treatment. A maximal increase of p21 mRNA in response to 1, 25-(OH)2D3 occurs after 48 h in ALVA-31 cells, and after 96 h in LNCaP cells. In contrast, the cell lines JCA-1 and TSU-Pr1 do not show any trends in regulation of p21 mRNA by 1, 25-(OH)2D3. The results presented in Fig. 3
demonstrate that 1, 25-(OH)2D3 treatment can affect the amount of p21 mRNA present in certain PC cell lines. In addition, a distinct correlation is evident between the up-regulation of p21 mRNA expression and growth sensitivity to 1, 25-(OH)2D3.
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, depicts a series of Western blots using protein lysates harvested from each of the four cell lines after exposure to 1, 25-(OH)2D3 and decorated with a p21 antibody. Fig. 4, A and B
, reveal that ALVA-31 and LNCaP cells up-regulate p21 protein levels in response to 1, 25-(OH)2D3. Quantitation of these signals revealed a maximal increase at 96 h, reaching 2.8-fold in ALVA-31 cells and 6.3-fold in LNCaP cells when compared with the 96-h ethanol vehicle control. These results with ALVA-31 are in contrast to that reported by Zhuang and Burnstein (23)
, who were unable to detect p21 expression in this cell line. The cell lines JCA-1 and TSU-Pr1 do not show any consistent trend in the regulation of p21 protein (Fig. 4, C and D)
, in agreement with our findings with p21 mRNA above. To control for loading error, each blot was stripped and reprobed for ß actin, indicating no significant differences (data not shown). These data indicate that 1, 25-(OH)2D3 is capable of inducing p21 protein expression in certain cell lines. In addition, a correlation is apparent between increased p21 protein expression and 1, 25-(OH)2D3 growth sensitivity.
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, 25-(OH)2D3 Is Abolished in ALVA-31 Cells Stably Transfected with Antisense p21 cDNA., 25-(OH)2D3 in PC cells, the data are only correlative. To determine whether p21 expression is necessary for 1, 25-(OH)2D3-mediated growth inhibition, we stably transfected the antisense p21 cDNA (ASp21) into the ALVA-31 cell line. Three clones were chosen, each of which expressed undetectable levels of p21 by Western blot analysis (data not shown). Fig. 5
illustrates the 1, 25-(OH)2D3-mediated growth response of parental ALVA-31 cells, ALVA-31 cells transfected with the LNCX vector only, and three ALVA-31 clones stably transfected with the p21 antisense cDNA. The graph illustrates that the parental ALVA-31 cells and the vector-transfected clone are growth inhibited by 1, 25(OH)2D3 in a dose-dependent fashion. However, the ASp21 clones were either insensitive or far less sensitive to hormone treatment. Differences in the percentage inhibition between the ASp21 clones and the control cell lines were statistically significant at each concentration of 1, 25(OH)2D3 tested (P < 0.05). The minimal amount of growth inhibition occurring in the antisense clones and the variation between clones is most likely attributable to the inability of antisense expression to block 100% of the p21 mRNA expression or induction. Consequently, it is plausible that small amounts of p21 are still being expressed in these clones. These findings are consistent with the initial hypothesis that p21 up-regulation by 1, 25-(OH)2D3 mediates the antiproliferative effects observed in the growth-sensitive PC cell lines. Because the clone Asp21#1 appeared the least sensitive to the growth inhibitory properties of amount of 1, 25-(OH)2D3, this clone was chosen for additional characterization.
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, 25-(OH)2D3 is the result of a decreased p21 level, it is also possible that this clone has lost its expression of functional VDRs. In an effort to ensure the presence of active VDRs, transient transfection experiments were performed using recombinant DNA containing the 24-hydroxylase VDRE joined to a luciferase reporter gene. We found that the fold induction of luciferase was similar after treatment with 1, 25-(OH)2D3 in the parental ALVA-31 cells, the vector-transfected ALVA-31 clone, and the ASp21#1 clone (4.4 ± 0.2, 4.5 ± 0.3, and 6.5 ± 0.3, respectively). This indicates that functional VDRs are present in the antisense p21-transfected clone.
ASp21#1 Is Incapable of Up-Regulating p21 in Response to Hormone Treatment.
As we have shown in Fig. 2
, baseline expression of p21 does not predict the sensitivity of any given cell line to the antiproliferative effects of 1, 25-(OH)2D3. Even relatively low levels of p21 can mediate this effect, as seen in the ALVA-31 cell line. Although the use of antisense cDNA oligomers is often effective at reducing the expression of specific proteins, it is unlikely that gene expression is completely extinguished. Therefore, it is possible that the ASp21#1 clone still expresses some baseline level of p21 mRNA. We wanted to determine whether any induction occurred in response to 1, 25-(OH)2D3 treatment. Fig. 6
shows the results of real-time quantitative RT-PCR for p21 mRNA in the parental ALVA-31 cell line, the vector-only transfected clone, and the ASp21#1 clone after treatment with 10-8 M 1, 25-(OH)2D3. Whereas the parental cells and the vector-only cells up-regulate the amount of p21 mRNA present after 1, 25-(OH)2D3 treatment, the levels were detectable but unchanged in ASp21#1. This implies that the induction of p21 mRNA by 1, 25-(OH)2D3, whether direct or indirect, is a required step in the growth inhibitory pathway of 1, 25-(OH)2D3 in this PC cell line.
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, 25-(OH)2D3 Do not Up-Regulate p21 after Hormone Treatment., 25-(OH)2D3 occurs within the ALVA-31 parental population, we isolated and expanded clones from that population. Four of the six resulting sublines demonstrated a similar degree of growth inhibition to the parental ALVA-31 population (
20%) in response to 10-8 M 1, 25-(OH)2D3 (data not shown). Two of the sublines, AL 2 and AL 7, were only growth inhibited by 5 and 10%, respectively. To determine whether these clones also up-regulate p21 protein levels in response to 10-8 M 1, 25-(OH)2D3, Western blot analyses were performed after 4 days of treatment. As shown in Fig. 7
, the growth-sensitive parental ALVA-31 cell line up-regulates p21 expression in response to 1, 25-(OH)2D3, whereas the two least growth-sensitive sublines show no change in p21 levels. Again, it is important to note the variation in basal p21 expression by each cell line and the lack of correlation of that level to 1, 25-(OH)2D3 growth sensitivity. The results in Fig. 7
suggest that the loss of regulation of p21 expression by 1, 25-(OH)2D3 is a naturally occurring phenomenon in a subset of ALVA-31 cells that also correlates with a reduced sensitivity to the antiproliferative effects of this hormone. These data additionally support our hypothesis that p21 up-regulation is critical for the growth inhibitory effects of 1, 25-(OH)2D3.
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, 25-(OH)2D3., 25-(OH)2D3, p21 levels were overexpressed in two cell lines that are otherwise incapable of up-regulating p21 in response to 1, 25-(OH)2D3 treatment. JCA-1 and TSU-Pr1 cells were transduced with adenoviral constructs that drive expression of either p21 or GFP (to control for any nonspecific effects of adenoviral gene transduction). The graph in Fig. 8
shows that both cell lines grow at a markedly slower rate when transduced with Ad-p21 than with Ad- GFP. Therefore, the additional p21 appears to be sufficient to inhibit the proliferation of these two cell lines even in the absence of 1, 25-(OH)2D3. Together, our results with JCA-1 and TSU-Pr1 suggest that the p21 signaling pathway is intact; however, it fails to be regulated by 1, 25-(OH)2D3.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 25-(OH)2D3 treatment clearly inhibits the proliferation of a variety of cell types (51)
. This antiproliferative effect can be attributed to the induction of apoptosis in some systems or the accumulation of cells in the G0/G1 phase of the cell cycle in others. At present, there is no consensus mechanism for this antiproliferative effect, and it appears that cell/tissue-specific mechanisms are operative. In agreement with previous reports (23
, 30)
, we were unable to detect the induction of apoptosis in any PC cell line after treatment with 1, 25-(OH)2D3 (data not shown). In the cell line LNCaP, the effect is attributable to the accumulation of cells in G0/G1 (23
, 30)
, although as we have shown in Table 1
and Fig. 1
, different experimental methods can greatly alter the magnitude of this response. Interestingly, there is also significant variation between laboratories regarding the basal percentage of LNCaP in G0/G1 in the control groups. Whereas we observed 70–80% in G0/G1, other groups have reported 45% (30)
and 57% (23)
using methods and plating densities within the ranges we tested. We can only speculate that these different basal levels of proliferation are because of the different passage numbers of LNCaP cells used. Our cultures were obtained directly from the originating lab and were used at early passage numbers (p 17–35), because older cultures display altered phenotypes that are accompanied by a more rapid rate of proliferation.
Flow cytometric studies with the cell line ALVA-31 have not revealed any significant changes in cell cycle distribution or apoptosis (23)
in agreement with our unpublished findings. Knowing that 1, 25-(OH)2D3 significantly reduces the growth of these cells in culture, this result seems counterintuitive. However, a careful review of the reported effects of p21 on cell proliferation provides insight into this phenomenon. p21 has largely been studied with respect to its inhibitory effects on cyclin-CDK complexes involved in cell cycle arrest during the G0/G1 phase of the cell cycle, such as cyclin D-CDK and cyclin E-CDK complexes (52)
. However, early experiments to assay the activity of p21 found it to be a universal inhibitor of CDKs (53)
. Furthermore, p21 mRNA in human fibroblasts demonstrates bimodal periodicity, with levels peaking in both G0/G1 and G2-M (54)
, thereby suggesting a role for p21 in the control of the cell cycle during each of these phase transitions. Therefore, it is possible that changes in p21 levels may control the rate of progression through the cell cycle without necessarily causing a measurable arrest in any particular phase. Our failure to detect changes in cell cycle distribution with ALVA-31 by flow cytometry are consistent with such a model.
Whereas others have attributed the lack of cell cycle arrest to an absence of p21 expression (23)
, we find that ALVA-31 cells do express both p21 mRNA and protein. More importantly, we demonstrated that these cells up-regulate p21 expression in response to 1, 25-(OH)2D3 treatment. Furthermore, we found that increased levels of p21 expression result in growth inhibition in two cell lines that do not respond to 1, 25-(OH)2D3. A newly published report with LNCaP cells additionally supports our proposed model. Boyle et al. (55)
provide evidence that 1, 25-(OH)2D3 up-regulates expression of insulin-like growth factor binding protein-3 in LNCaP cells, which is critical for the up-regulation of p21 and subsequent growth inhibition. These important results, together with our findings, suggest that the p21 pathway commonly mediates the antiproliferative effects of 1, 25-(OH)2D3 in prostatic epithelial cells.
Recent studies from our laboratory have revealed convincing and important data in regard to the true origins of several cell lines that were thought to be unique and of prostatic origin. The cell line ALVA-31 is most likely to be a subline of PC-3 resulting from cross-contamination in the originating laboratory. This conclusion is based on the results of cytogenetic and mutational analyses, DNA profiling, and multiplex-fluorescence in situ hybridization (56 , 57) . Of interest is that the ALVA-31 cell line demonstrates significantly different phenotypic properties than its parent cell line, PC-3. For this reason, the ALVA-31 cell line should remain useful, because it may reflect the natural heterogeneity that occurs within prostate tumors from the same patient. Therefore, we don’t believe that these findings diminish the value of our data in the current study. However, the recent genetic analyses of the cell lines TSU-Pr1 and JCA-1 will have a greater impact in the field of PC research. These results reveal not only that JCA-1 shares a common origin with TSU-Pr1 (57) , but that both of these lines are very likely to be sublines of the T24 bladder carcinoma cell line (58) . Whereas our studies with the sublines of ALVA-31 suggest that this loss of regulation can occur in PC cells, additional analyses are necessary to determine whether the PC cell lines PC-3 and DU-145 behave like JCA-1 and TSU-Pr1, because they are also relatively resistant to the antiproliferative effects of this hormone.
In addition to its well-known role in p53-dependent checkpoint control of cell cycle progression, p21 has been found to be involved in mediating a p53-independent growth arrest associated with terminal differentiation (59) . Other functions attributed to p21 include: cell cycle arrest after DNA damage; senescence-associated growth arrest; inhibition of SAP kinase activity; cyclin-CDK assembly and activation; and both inhibition and induction of apoptosis (60) . In view of the numerous contrasting functions of p21, it is likely that the stoichiometry of p21 is important. For example, p21 may assist in cyclin-CDK assembly and activation of kinase activity at low concentrations, thereby inhibiting cyclin-CDK dissociation. However, at higher concentrations p21 acts to inhibit CDK activity, resulting in cell cycle arrest.
Our investigation focused on the role of p21 in mediating the growth inhibitory effect of 1, 25-(OH)2D3 in PC cell lines. Neither the basal expression level of p21 mRNA nor p21 protein was found to be associated with the degree of growth inhibition by 1, 25-(OH)2D3 treatment. Thus, we conclude that the absolute level of p21 in the cell does not determine growth response to 1, 25-(OH)2D3. Instead, our studies consistently show that p21 induction in response to 1, 25-(OH)2D3 correlates with a subsequent decrease in cell growth. Increasing the amount of p21 protein in the cell (in proportion to other cell cycle regulatory proteins) may be the primary factor controlling 1, 25-(OH)2D3-mediated antiproliferative effects. Therefore, we propose that the change in stoichiometry of p21 after exposure to 1, 25-(OH)2D3 controls cell proliferation. Experiments are now underway in our laboratory to explore ratios of p21 to other cell cycle mediators.
The regulation of p21 in response to 1, 25-(OH)2D3 treatment suggests its involvement in mediating the observed growth inhibitory effect. Additional evidence confirms an active role for p21 in some cell lines. ALVA-31 cells stably transfected with the antisense p21 construct are no longer able to up-regulate either their p21 mRNA or their p21 protein and no longer respond to the antiproliferative effect of 1, 25-(OH)2D3 treatment. The presence of functional VDRs in these clones was confirmed using a 24-hydroxylase VDRE in transient transfection analysis. Therefore, these results indicate that p21 up-regulation is a necessary prerequisite to growth inhibition of the ALVA-31 PC cell line by 1, 25-(OH)2D3.
Experiments using adenovirus to overexpress p21 helped to confirm our hypothesis that a change in the stoichiometry of p21 can directly inhibit growth of cell lines that are normally resistant to the growth inhibitory effects of 1, 25-(OH)2D3. Overexpression of p21 in JCA-1 and TSU-Pr1 cells, independent of 1, 25-(OH)2D3 treatment, caused the cells to grow at a slower rate. Similar antiproliferative effects of increased p21 expression have been observed in several other cell types indicating that this may be a general mechanism.
The expression of p21 has also been examined in tissue sections of prostatic carcinomas. Shiraishi et al. (61) found that 14/66 (21%) of carcinomas examined showed p21-positive nuclear staining from 1 to >50% of malignant cells. Aaltomaa et al. (62) also found that 73% (156/213) of prostate carcinomas contained focal areas of p21-positive cells. In addition, their results indicate that p21 expression is correlated with high Gleason score, DNA aneuploidy, high S phase fraction, bcl-2 expression, and cyclin D expression. It has also been suggested that the expression of p21, cyclin D1, and bcl-2 are related to androgen actions and could be relevant to the progression of PC to a hormone refractory state (63) . However, our findings suggest that increased expression of p21 should have an antiproliferative effect. It is possible that the positive immunostaining for p21 seen by others is the result of a stoichiometric shift in several regulators that ultimately favors proliferation. It is also possible that mutations of p21 such as those described in urothelial carcinomas (64) could be present and lead to elevated levels of nonfunctional p21. We are currently investigating these possibilities in carcinomas from prostatectomy specimens.
In conclusion, we propose a model in which 1, 25-(OH)2D3 treatment of PC cell lines causes an increase in p21 mRNA levels leading to an increase in p21 protein levels. The increased p21 levels are responsible for a gradual slowing of the cell cycle and/or the accumulation of cells in G0/G1 resulting in the antagonistic effect of 1, 25-(OH)2D3 on PC cell growth seen in vitro. Taken together, our findings suggest that the ability of a cell to regulate its p21 expression plays an essential role in this system. Additional studies are currently being conducted in our laboratory to determine the molecular pathways involved in the 1, 25-(OH)2D3 regulation of p21 expression and in the apparent failure of this pathway in selected cell lines. Such information should eventually be important in defining why some patients respond to 1, 25-(OH)2D3 and others do not in an ongoing clinical trial through the University of Colorado Health Sciences Center and may ultimately assist in our efforts to use this compound therapeutically.
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FOOTNOTES |
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1 Supported in part by USPHS Grant CA 53520 (to G. J. M.) and by USPHS Grant CA 46934 to the University of Colorado Cancer Center through the Cytogenetics and Quantitative PCR Core Laboratories. 
2 To whom requests for reprints should be addressed, at University of Colorado Health Sciences Center, Department of Pathology, Box B-216, 4200 East Ninth Avenue, Denver CO 80262. Phone: (303) 315-8449 or (303) 315-4567; Fax: (303) 315-6761; E-mail: Tammy.Hedlund{at}uchsc.edu
3 The abbreviations used are: PC, prostate cancer; 1, 25-(OH)2D3, 1, 25-dihydroxy vitamin D3; VDR, vitamin D receptor; CDK, cyclin-dependent kinase; VDRE, vitamin D response element; FBS, fetal bovine serum; RT-PCR, reverse transcription-PCR; GFP, jellyfish green fluorescent protein.
4 After the original submission of this manuscript, evidence has accumulated in regard to the true origins of several supposed PC cell lines. This includes ALVA-31, TSU-Pr1, and JCA-1 cells; see "Discussion."
Received 11/22/00. Accepted 7/31/01.
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