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,25-Dihydroxyvitamin D3 in Human Prostate Cancer Cell Line LNCaP Involves Reduction of Cyclin-Dependent Kinase 2 Activity and Persistent G1 Accumulation
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33136
Address all correspondence and requests for reprints to: Kerry L. Burnstein, Department of Molecular and Cellular Pharmacology (R-189), University of Miami School of Medicine, P.O. Box 016189, Miami, Florida 33101. E-mail: kburnste{at}molbio.med.miami.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-Dihydroxyvitamin D3 (1,25 D), the most active
metabolite of vitamin D3, exerts antiproliferative and
prodifferentiating effects on some human prostate cancer cell lines. We
previously reported an inverse relationship between functional vitamin
D receptor (VDR) levels and antiproliferative response to 1,25 D in two
human prostate cancer cell lines, LNCaP and ALVA 31. Although LNCaP
cells are far more sensitive to growth inhibition by 1,25 D than ALVA
31 cells, LNCaP express approximately half the number of VDR as ALVA
31. Two other human prostate cancer cell lines studied, PC3 and DU145,
express lower levels of functional VDR and are relatively insensitive
to growth inhibition by 1,25 D. In this report, we investigated
potential mechanisms of the variable antiproliferative activity of 1,25
D. In PC3 cells stably expressing VDR [PC3(VDR)] at levels comparable
to LNCaP, 1,25 D treatment resulted in only moderate growth inhibition.
These results further support the contention that VDR expression,
although required, is not sufficient for maximal growth suppression by
1,25 D, as is exhibited by LNCaP cells. We did not detect 1,25
D-mediated DNA fragmentation after 4 days of 1,25 D treatment in either
LNCaP or ALVA 31 cells. This result suggests that variability in 1,25 D
sensitivity does not derive from differences in the capacity of these
cells to undergo apoptosis in response to 1,25 D. Flow cytometry of
propidium iodine-stained cells revealed that 48 h 1,25 D treatment
of LNCaP cells resulted in a 2-fold decrease of cells in G2/M plus S
phases and accumulation of LNCaP cells in the G1/G0 phase. This effect
persisted for 72 h after 1,25 D removal. In contrast, 1,25 D did
not significantly alter the cell cycle distribution of ALVA 31 or
PC3(VDR) cells. Consistent with accumulation of cells in G1/G0, 1,25 D
treatment of LNCaP cells resulted in decreased retinoblastoma protein
phosphorylation, repressed E2F transcriptional activity, increased
levels of the cyclin-dependent kinase (CDK) inhibitor
p21WAF1, CIP1, and decreased CDK2 activity.
However, p21 messenger RNA levels were not altered, suggesting
translational or posttranslational regulation of p21 by 1,25 D. In
contrast, p21 was not detected in ALVA 31 or PC3(VDR) and was not
induced by 1,25 D, consistent with the failure of 1,25 D to influence
cell cycle distribution in these cells. These results suggest that
variability in sensitivity to the antiproliferative effects of 1,25 D
among prostate cancer cells is dependent, at least in part, on the
integrity of the retinoblastoma pathway and in particular on p21
expression and 1,25 D regulation of CDK2 activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 25-dihydroxyvitamin D3
(1,25 D), inhibits proliferation and induces differentiation of a
variety of normal and malignant cells (1, 2, 3). Based on epidemiological
data, Schwartz and Hulka (4) hypothesized that vitamin D maintains the
differentiated phenotype of prostatic cells, and that vitamin D
deficiency may be a risk factor for prostate cancer mortality (5, 6).
Subsequent laboratory studies demonstrated that 1,25 D inhibits the
growth of some established human prostate cancer cell lines as well as
primary epithelial cultures derived from benign and cancerous prostatic
tissue (7, 8, 9). However, the mechanism(s) of this growth inhibition and
the basis for the variability in antiproliferative effects of 1,25 D in
prostate cancer cells is not understood. Vitamin D action is mediated primarily through binding of 1,25 D to the intracellular vitamin D receptor (VDR), a member of the steroid/thyroid hormone receptor superfamily, which functions as a ligand-activated transcription factor (10). The VDR forms heterodimers with another member of this family, the retinoid X receptor (RXR), and regulates gene expression through binding to DNA sequences termed vitamin D response elements (VDREs) (11). We and others have shown that the lack of antiproliferative effects of 1,25 D in some human prostate cancer cell lines is associated with low levels of functional VDR (12, 13). Transfection of a VDR complementary DNA (cDNA) expression vector into the relatively 1,25 D-insensitive human prostate cancer cell lines PC3, DU145, and JCA-1 results in moderate growth inhibition by 1,25 D (12, 13). Blocking the expression of VDR in the human prostate cancer cell line ALVA 31 abolishes the growth inhibition by 1,25 D (14). These data support the hypothesis that the antiproliferative effect of 1,25 D in human prostate cancer cells is mediated by VDR. However, we demonstrated that LNCaP and ALVA 31 cells exhibit an inverse relationship between VDR levels and antiproliferative response to 1,25 D (13). Although ALVA 31 cells have almost twice as many VDRs as LNCaP, LNCaP are substantially more sensitive to growth inhibition by 1,25 D than ALVA 31 cells. VDRs in both LNCaP and ALVA 31 cells are transcriptionally active as demonstrated by reporter gene assays using two different VDREs (13). These results suggest that functional VDRs, although necessary, are not sufficient for maximal antiproliferative effects of 1,25 D on human prostate cancer cell lines.
Different mechanisms have been identified for 1,25 D inhibition of cell proliferation. 1,25 D induces morphological and biochemical markers of apoptosis (programed cell death) in MCF-7 human breast cancer cells (15). In a variety of human leukemic cells, 1,25 D induces G1 cell cycle arrest (reviewed in Ref.16). This effect on cell cycle correlates with 1,25 D-induced expression of cyclin-dependent kinase (CDK) inhibitors (CKIs) including p21, p27, and members of the INK4 family in the myelomonocytic cell line U937 (17). The induction of p21 by 1,25 D in U937 cells may be mediated by a VDRE identified in the human p21 promoter and also through posttranscriptional mechanisms (17).
We investigated the possible mechanisms of the antiproliferative effect of 1,25 D in human prostate cancer cell lines. We did not detect 1,25 D-mediated DNA fragmentation in LNCaP cells, ALVA 31 cells, or in PC3 cells stably expressing VDR [PC3(VDR)]. Thus, differences in the capacity of cells to undergo apoptosis do not appear to underlie the variable growth inhibition by 1,25 D in these cell lines. 1,25 D caused LNCaP cells to accumulate in G1/G0 even after 1,25 D removal. Whereas 1,25 D did not significantly influence cell cycle distribution of ALVA 31 or PC3(VDR) cells. These effects of 1,25 D appear to be mediated through the retinoblastoma (Rb) protein pathway, as 1,25 D-treated LNCaP cells expressed increased levels of the hypophosphorylated form of Rb. Consistent with its effects on Rb, 1,25 D repressed E2F transcriptional activity and reduced CDK2 activity. 1,25 D treatment resulted in up-regulation of the CKI p21 in LNCaP cells; however, p21 expression was not detected in the presence or absence of 1,25 D in ALVA 31 cells or PC3(VDR) cells. Thus, maximal growth sensitivity of human prostate cancer cells to 1,25 D is associated with accumulation of cells in G1/G0 and induction of the CKI p21, but not with apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-Dihydroxyvitamin D3 was purchased from
BIOMOL Research Labs. (Plymouth Meeting, PA). In situ
end-labeling kit was purchased from Trevigen (Gaithersburg, MD).
Propidium iodide was purchased from Sigma (St. Louis, MO). RNase A was
obtained from Boehringer Mannheim (Indianapolis, IN).
[26,27-methyl-3H]1,25-Dihydroxyvitamin D3
and [14C]chloramphenicol were obtained from Dupont-NEN
(Boston, MA).
Cell culture, growth curves, and flow cytometry
The human prostate carcinoma cell lines LNCaP.FGC [LNCaP
(ATCC cat. no. CRL1740; batch F-11701)] and PC3 (ATCC cat.
no. CRL 1435; batch F-11154) were obtained from American Type Culture
Collection (Rockville, MD). The human prostate carcinoma cell line ALVA
31 (18) was generously provided to the University of Miami Cell Culture
Core Facility by Drs. Stephen Loop and Richard Ostenson (Department of
Veterans Affairs Medical Center, Tacoma, WA). All cell lines were
routinely cultured in RPMI-1640 containing 100 IU/ml penicillin, 100
µg/ml streptomycin, 2 mM L-glutamine (GIBCO BRL,
Gaithersburg, MD), and 10% FBS. Briefly, LNCaP is an androgen
receptor-positive, androgen-sensitive human prostate cancer cell line
(19); PC3 and ALVA 31 cells do not express detectable levels of
androgen receptor and are not growth regulated by androgen in
vitro (18, 20). ALVA 31 cells are derived from a
well-differentiated stage B2 prostate adenocarcinoma (18).
For cell proliferation studies, cells were plated at a density of 20,000/well of six-well cluster dishes in RPMI-1640 media supplemented with 10% FBS. The cells were treated with vehicle or 10 nM 1,25 D 24 h after plating and were trypsinized and counted using a hemacytometer on days 1, 2, 3, 4, 6, and 8 after treatment. The media and 1,25 D were replaced on day 4 for cells cultured to day 6 and day 8. Cell viability was assessed by trypan blue exclusion and found to be >95%. All experiments were performed in duplicate. LNCaP and ALVA 31 were grown in serum-supplemented media, because these cells proliferate relatively rapidly in this medium with very few floating cells (<1%). LNCaP cells did not reach confluence by 8 days, whereas control ALVA 31 cells became confluent after 6 days in culture. The proliferation data were plotted on a common log scale and fit by linear regression using the formula: y = log(Ct/C0) = (log 2/tD)t, where Ct and C0 represent the cell number at time point t or that at the starting point, respectively; tD stands for doubling time. The doubling time was calculated by tD = Log2/K (days), where K is the slope of the regression line. Goodness of fit (r2) was obtained using the Sigmaplot program (Jandel Corp., San Rafael, CA).
For fluorescence-activated cell sorting (FACS) analysis, cells were plated at a density of 100,000 cells per 100-mm dish in RPMI-1640 supplemented with 10% FBS. Twenty-four hours after plating, the cells were treated with vehicle or 10 nM 1,25 D and trypsinized after the indicated time. The cells were washed twice with ice-cold PBS, fixed by drop-wise addition of 70% ethanol at approximately 1 x 106 cells/ml, and incubated at 4 C overnight with constant agitation. Thirty minutes before flow cytometry analysis, the cellular double stranded nucleic acids were stained with propidium iodine (50 µg/ml). RNase A (100 U/ml) was included to degrade double stranded RNA. Propidium iodine fluorescence was obtained using linear amplification with doublet discrimination. Five thousand forward scatter gated events were collected per sample. Data were analyzed by the Cellquest program (Becton-Dickinson, San Jose, CA). To test the persistence of 1,25 D-mediated cell cycle effects, the cells were treated with 10 nM 1,25 D for 48 h. 1,25 D was then removed by washing the cell monolayers three times with 1x PBS at 37 C. Removal of 1,25 D was tested by subsequent VDRE reporter gene assay (described below). After removal of 1,25 D, the cells were cultured in complete media without 1,25 D; harvested after 12, 24, 48, and 72 h; and subjected to FACS analysis.
Tdt-mediated dUTP nick end labeling (TUNEL) assay
Cells were plated and treated as described above for the growth
curve experiments and fixed using 4% paraformaldehyde on days 1, 2, 3,
and 4. TUNEL assay was conducted following the instructions provided by
the manufacturer (Trevigen, Gaithersburg, MD). Briefly, cells were
permeabilized using proteinase K and endogenous peroxidase was
inhibited by incubation in 2% H2O2 for 5 min.
The cells were then incubated with labeling mix containing terminal
deoxynucleotide transferase (Tdt) and biotinylated nucleotide mix for
2 h at 37 C in a humidified CO2 incubator. Cells were
washed and incubated with streptavidin-horseradish peroxidase conjugate
followed by washing and color reactions to detect DNA fragmentation,
which appeared as dark blue-stained nuclei. Cells were counterstained
with red counterstain B. Cells were visualized using a Nikon microscope
(model HB-10101AF) (Nikon Corp., Tokyo, Japan), and data were
quantified by counting dark blue-stained nuclei from 5–10 randomly
chosen fields.
Western blotting
Twenty four hours after plating, the cells were treated
with vehicle or 10 nM 1,25 D for the times indicated. Cells
were then trypsinized, washed twice with ice-cold 1x PBS, and lysed in
50 mM Tris, pH 7.4, 250 mM NaCl, 2
mM EDTA, 1% Nonidet P-40, 1 mM
phenylmethylsulfonylfluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 50 mM NaF, and 0.1 mM
NaVO4. After a 5-min incubation on ice, the lysate was
centrifuged, and the supernatant (cell extract) was collected. The
protein concentration was determined by Bio-Rad DC Protein
assay (Bio-Rad, Hercules, CA) according to the manufacturer’s
instructions. Cell extract proteins (100–200 µg) were subjected to
SDS-PAGE and transferred to nitrocellulose membrane filters. Standard
curves were set up to establish the linearity of the assay. Filters
were processed for immunoblotting using standard procedures. Briefly,
filters were incubated in blocking solution (5% dry milk in 1x TBS)
followed by incubation with primary antibody for 3 h. The
following primary antibodies were used at 1 µg/ml: p21, CDK5, and
cyclin D1, cyclin D2, and cyclin E antibodies from Oncogene Research
Products (Cambridge, MA); CDK4, CDK6, and p53 antibodies from Santa
Cruz Biotechnology (Santa Cruz, CA); Rb and CDK2 antibodies from
Pharmingen (San Diego, CA); or 0.5 µg/ml actin antibody (Boehringer
Mannheim). After washing, the blot was incubated with horseradish
peroxidase-conjugated secondary antibody, and proteins were visualized
using the ECL system (Amersham, Buckinghamshire, UK) following the
supplier’s instructions. Data were quantified using NIH Image 1.60
(NIH, Bethesda, MD).
Northern blotting
Total RNA was isolated using TRIzol reagent (GIBCO, Grand
Island, NY) according to the manufacturer’s instructions. Thirty
micrograms of total RNA was denatured in dimethyl sulfoxide and
glyoxal. Denatured RNA was electrophoresed in a 1% agarose gel and
transferred to a Nytran membrane (Schleicher & Schuell, Keene, NH).
Northern blotting was conducted using standard procedures. Blots were
hybridized with 32P-labeled p21 cDNA probe (from Dr. David
Beach, Cold Spring Harbor Laboratory, NY). After washing with 6x SSPE,
0.1% SDS for 15 min at room temperature, twice with 1x SSPE, 0.5%
SDS for 15 min at 68 C, and once with 0.1x SSPE, 0.1% SDS for 1
h at 68 C, the membrane was exposed to X-ray film at -80 C using an
intensifying screen. After exposure, the membrane was stripped and
hybridized with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA
probe to normalize for RNA loading. Data were quantified using NIH
Image 1.60.
Establishment of PC3 cells stably expressing VDR
The VDR cDNA expression vector (pRc-CMV-VDR) (provided by
Dr. Leonard Freedman, Memorial Sloan-Kettering Cancer Center, New York,
NY) was transfected into PC3 cells using the calcium phosphate method.
Cells were then plated in 96-well plates at limiting dilution and
cultured in media containing G418 (Geneticin, GIBCO BRL) at 350 µg/ml
(active drug concentration). Two to four weeks later, G418-resistant
cells were replated and screened for VDR expression by single-point
radioligand binding assay and reporter gene assays (described below).
PC3 cells transfected with the pcDNA3 vector alone [PC3 (neo)] were
selected with G418. G418-resistant cells were pooled and used as a
control. PC3(VDR) and PC3(neo) were maintained continuously in 350
µg/ml G418.
Reporter gene assays
The construction of the reporter plasmid MOPVDREtkCAT, which
contains two tandem copies of the mouse osteopontin (MOP) VDRE linked
to the thymidine kinase (tk) promoter and chloramphenicol
acetyltransferase (CAT) gene, was described previously (13).
MOPVDREtkCAT was transfected into PC3(VDR) clones using the calcium
phosphate method. E2F reporter gene constructs pE2wtCAT and
pE2(-64/-60, -45/-36)CAT (21, 22) were provided by Dr. John Brady
(National Cancer Institute, Bethesda, MD) and Dr. Mary R. Loeken
(Joslin Diabetes Center, Harvard Medical School, Boston, MA). A p21
promoter-reporter gene construct was obtained from Dr. Bert Vogelstein
(Johns Hopkins University, Baltimore, MD) (23). Cytomegalovirus
(CMV)-ß-gal, which encodes the ß-galactosidase gene driven by the
CMV promoter, was included in all transfections to normalize for
differences in transfection efficiency. The cells were treated with
vehicle or 10 nM 1,25 D, then harvested approximately
40 h later. Cell extracts were prepared for analysis of
ß-galactosidase and CAT activity. ß-galactosidase assays were
performed as described (24). Cell extracts containing equivalent
amounts of ß-galactosidase activities were used for analysis of CAT
activity using an adaptation of the method of Gorman et al.
(25). The percent conversion of [14C]chloramphenicol to
acetylated forms on thin-layer chromatograms was quantified using a
Molecular Dynamics PhosphorImager and ImageQuant software (Sunnyvale,
CA).
Radioligand binding assay
Soluble cell extracts (cytosols) were prepared by a modification
of the method of Baker et al. (26). Near confluent 100-mm
dishes of cells were harvested and homogenized using a Dounce
homogenizer (Wheaton, Millville, NJ) (pestle B) in TK0.3DE buffer [10
mM Tris (pH 7.4), 300 mM KCL, 1 mM
dithiothreitol, 1.5 mM EDTA, and 10 mM sodium
molybdate] containing protease inhibitors (1 µg/ml leupeptin, 2
µg/ml pepstatin, and 0.2 mM
phenylmethylsulfonylfluoride). The homogenates were then centrifuged at
210,000 x g for 35 min at 4 C. The supernatants were
collected and used for binding studies. The protein concentration was
determined by Bio-Rad DC Protein assay according to
manufacturer’s instruction. Cytosols (200 µl containing 1 mg/ml
protein) were incubated with 1 nM [3H]1,25 D
with or without 500-fold excess of radioinert 1,25 D. Bound and free
1,25 D were separated by the hydroxylapatite method (27). Specific
binding was calculated by subtracting nonspecific binding from total
binding. Experiments were performed in duplicate.
Immunoprecipitation and in vitro kinase assay
Cells were plated as for flow cytometry and growth curves. The
cells were harvested at 24 and 48 h after 1,25 D treatment and
lysed. Four hundred micrograms cell lysate proteins were incubated with
1 µg CDK2 polyclonal antibody for 1 h at 4 C with agitation.
Thirty microliters protein G plus-agarose (Santa Cruz Biotechnology)
were then added to each tube and incubated for 3 h. The immune
complexes were collected by centrifugation. After washing three times
with lysis buffer and three times with kinase assay buffer, 30 µl
kinase assay mix containing 2 µg substrate (histone H1) (Boehringer
Mannheim), 25 µM ATP, and 10 µCi
[
32P]ATP were added, and the complexes were incubated
for 30 min at 30 C. The reactions were stopped by addition of 2x
sample buffer. After 3 min boiling, the reactions were subjected to
standard SDS-PAGE and transferred to nitrocellulose membranes.
Phosphorylation of substrate was visualized by autoradiography. After
autoradiography, membranes were subjected to Western blotting using
anti-CDK2 antibody to determine the amount of CDK2 present in the
immunocomplexes. Phosphorylation of histone H1 was quantified using a
Molecular Dynamics PhosphorImager and ImageQuant software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B). The
doubling time of LNCaP cells increased from 28 ± 1.6 h to
61 ± 0.6 h with 1,25 D treatment (Fig. 1A). 1,25 D treatment
slightly but significantly (P < 0.05) increased the
doubling time of ALVA 31 cells from 22 ± 0.5 h to 24 ±
0.5 h (Fig. 1B). Because ALVA 31 cells express almost twice as
many VDR as LNCaP cells (13), the modest antiproliferative effect of
1,25 D on ALVA 31 cell proliferation compared with that observed in
LNCaP cells indicates that VDR levels do not strictly correlate with
growth sensitivity to the hormone.
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). These assays revealed that both
clones expressed functional VDR, and that CAT induction (25- to 35-fold
induction) was roughly proportional to VDR levels. However, despite the
presence of functional VDR at approximately the same level as LNCaP,
1,25 D treatment resulted in a relatively modest (1.4-fold) increase in
doubling time (from 38 h to 52 h) in PC3(VDR) (3B2 clone)
(Fig. 2B). Similar results were obtained for clone 2B12 (data not
shown). Under the same conditions, 1,25 D caused a 2.2-fold increase in
doubling time of LNCaP cells (Fig. 1A
). These results suggest that
functional VDR, although necessary, is not sufficient for the profound
antiproliferative effect of 1,25 D observed in LNCaP cells.
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).
By contrast, we did not detect DNA fragmentation in LNCaP cells in the
absence or presence of 1,25 D after 2 days (Fig. 3, B and C) or 4 days
(Fig. 3, D and E). Similarly, DNA fragmentation in LNCaP cells was not
evident after 6 days of 1,25 D treatment (data not shown), but by 8
days both vehicle-treated and 1,25 D-treated LNCaP cells exhibited some
DNA fragmentation (data not shown). Neither PC3(VDR) nor ALVA 31
exhibited 1,25 D-mediated DNA fragmentation after 4 days (data not
shown). Therefore, differences in 1,25 D growth sensitivity in LNCaP
compared with ALVA 31 and PC3(VDR) cells are unlikely to involve
apoptotic mechanisms.
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. We found that 1,25 D caused LNCaP
cells to accumulate in the G1/G0 phase of the cell cycle (Fig. 4),
consistent with the recently published findings of Weigel and
colleagues (30). Our results from two experiments at the 24-h and three
experiments at the 48-h time points show that the percentage of LNCaP
cells in G2/M + S phases decreased from 43% to 22% after 48 h
treatment with 10 nM 1,25 D (Fig. 5). We examined the cell cycle
distribution of LNCaP cells at 3, 4, 6, and 8 days of 1,25 D treatment
and observed that the percent of cells in S phase decreased to 6
± 1% after 3 days and progressively decreased to 4.5 ± 0.5%
after 8 days (data not shown). By 8 days, 86% of 1,25 D-treated LNCaP
cells were in G1/G0 (data not shown). Thus, the high degree of 1,25
D-mediated growth inhibition exhibited by LNCaP cells is likely to be
due, at least in part, to inhibition of cellular progression to S
phase. The cell cycle distribution of ALVA 31 cells was not
significantly altered by 1,25 D at 48 h (Figs. 4 and 5) or after 4
days treatment (data not shown), nor was cell cycle distribution of
PC3(VDR) affected by 1,25 D (data not shown). These data suggest that
the differential antiproliferative effects of 1,25 D in prostate cancer
cell lines may be due to varying susceptibility of cells to accumulate
in the G1/G0 phase of the cell cycle in response to 1,25 D.
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shows that cell cycle
distribution of LNCaP cells did not change significantly after 72
h incubation in the absence of 1,25 D. This result indicates that 1,25
D-mediated effects on cell cycle in LNCaP cells persist for up to 3
days after removal of 1,25D.
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). E2F transcriptional
activity was monitored in LNCaP cells using two E2F reporter gene
constructs. pE2wtCAT consists of the E2A promoter/enhancer sequence
bearing two E2F binding sites linked to CAT and
pE2(-64/-60,-45/-36)CAT in which the two E2F binding sites are
mutated (21). As shown in Fig. 6B, E2F transcriptional activity was
decreased in 1,25 D-treated LNCaP cells. Use of the reporter plasmid
containing mutated E2F binding sites resulted in no reporter gene (CAT)
expression, indicating that the CAT activity observed in LNCaP cells
was dependent on activated E2Fs. This result is consistent with 1,25
D-induced G1/G0 cell cycle accumulation of LNCaP cells. 1,25 D did not
repress E2F transcriptional activity in ALVA 31 cells (data not
shown).
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, A and B). This result suggests that
1,25 D may inhibit the growth of LNCaP cells in part by inducing
expression of p21. In contrast, p21 was not expressed in ALVA 31 or
PC3(VDR) cells (Fig. 7A) in the presence or absence of 1,25 D
treatment, consistent with the failure of 1,25 D to alter cell cycle
distribution in these cells. 1,25 D did not regulate the levels of
CDK2, CDK4, CDK5, and CDK6 or cyclins D1, D2, or E after 48 h
treatment (Fig. 7, A-C). Levels of the tumor suppressor protein p53
also were not significantly altered by 1,25 D in LNCaP cells (Fig. 7D).
Consistent with the lack of p21, p53 protein, a transcriptional
up-regulator of p21 (31), was not detected in PC3(VDR) or ALVA 31 (Fig. 7D).
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). 1,25 D did not alter the steady
state levels of p21 mRNA in LNCaP cells (Fig. 8, A and B). To test this
observation further, we transfected LNCaP cells with a p21
promoter-luciferase reporter plasmid containing 2.6 kb of the human p21
promoter linked to a luciferase gene. Luciferase activity was measured
in 1,25 D-treated and vehicle-treated cells; 1,25 D did not induce p21
promoter activity in LNCaP cells (data not shown). These data suggest
that 1,25 D may induce p21 protein levels through translational or
posttranslational mechanisms.
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, upper
panel). The filters were than processed for Western blot
analysis to show that comparable levels of CDK2 protein were
present in the treatment groups (Fig. 9, lower panel). 1,25
D did not affect CDK2 activity in ALVA 31 cells (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), differential growth sensitivity to 1,25 D is not likely to
be due to VDR or RXR mutation or to differences in 1,25 D
metabolism. Together these studies indicate that expression of
functional VDR is necessary, but not sufficient, for maximal growth
inhibition by 1,25 D. Moreover, these results predict that 1,25 D
target genes involved in cellular proliferation and/or cell death may
be differentially expressed and/or regulated in human prostate cancer
cell lines. The mechanism of growth inhibition of LNCaP cells by 1,25 D involves hormone-induced accumulation of cells in the G1/G0 phase of the cell cycle. Similar results were reported recently by Blutt et al. (30). In addition to this initial finding, we have assessed the possible roles of cell cycle control and apoptotic mechanisms in the differential effect of 1,25 D on the growth of several prostate cancer cell lines. No significant change in cell cycle distribution was observed in ALVA 31 or PC3(VDR) cells in response to 1,25 D. These results indicate that the variability of antiproliferative effects of 1,25 D in human prostate cancer cells is at least partially due to differences in the capacity of these cells to accumulate in the G1/G0 phase of the cell cycle in response to 1,25 D. Failure of ALVA 31 and PC3(VDR) cells to accumulate in G1/G0 after 1,25 D treatment suggests that other mechanisms must be responsible for the modest antiproliferative effects of 1,25 D in these cells. 1,25 D treatment of ALVA 31 and PC3(VDR) may result in small, but equivalent inhibition of all phases of the cell cycle that does not alter the percentage of cells in any particular phase. Such a general suppressive effect of 1,25 D on all cell cycle phases might decrease proliferation rate by lengthening the cell cycle. We did not observe 1,25 D-mediated DNA fragmentation in 1,25 D-sensitive cells, LNCaP, or in the less-sensitive cells, ALVA 31 and PC3(VDR), under conditions that are sufficient for growth inhibition. Thus, the profound differences in the extent of 1,25 D-mediated growth inhibition of prostate cancer cell lines are unlikely to be due to apoptotic mechanisms. Together, these findings predict that 1,25 D might regulate specific G1 regulatory proteins in LNCaP but not in ALVA 31 or PC3(VDR) cells.
Our observation that G1/G0 accumulation in response to 1,25 D was persistent for several days after 1,25 D removal is consistent with reports that 1,25 D promotes differentiation in LNCaP cells. Miller et al. (32) and Skowronski et al. (7) showed that 1,25 D increases expression of prostate-specific antigen, a differentiation marker for prostate secretory epithelial cells. Furthermore, Peehl et al. (8) reported that very short (2 h) exposure of primary prostate cultures to 1,25 D resulted in irreversible growth inhibition.
Regulation of cellular progression from G1 to S phase is one of
the most critical steps in cell cycle control, and the step most
frequently altered in cancers (31, 33). The action of G1 cyclins and
their specific CDK binding partners governs phosphorylation of Rb. The
hyperphosphorylation of Rb results in progression of cells from G1 to S
phase. Both LNCaP cells and PC3 cells express functional Rb (34). Thus,
alterations in Rb are unlikely to underlie the differential 1,25
D-mediated growth inhibition exhibited by LNCaP and PC3(VDR) cells.
1,25 D did not affect the phosphorylation of Rb in ALVA 31 cells (Fig. 6A); this finding is consistent with failure of ALVA 31 cells to
accumulate in G1/G0 phase in response to 1,25 D. 1,25 D was found to
up-regulate several CKIs in the myelomonocytic cell line U937.
Regulation of these inhibitors, particularly p21 and p27, appears to be
sufficient for the differentiation promoting effects of 1,25 D in these
cells (17). 1,25 D up-regulation of p27 in HL60 cells also correlates
with G1 arrest and the differentiating effects of 1,25 D (35). We found
that 1,25 D up-regulated p21 in LNCaP cells. Although up-regulation of
p21 by 1,25 D was modest, we observed substantial reduction in CDK2
activity in 1,25 D-treated LNCaP cells (Fig. 9). The role of p21 in
this 1,25 D-mediated decrease in CDK2 activity is currently being
investigated. Neither ALVA 31 nor PC3(VDR) cells expressed detectable
p21 or p53 protein (Fig. 7). The lack of p21 in ALVA 31 and PC3(VDR)
cells may be due to the absence of p53 expression, because p53 is known
to induce p21 (23). A significant association between p53 abnormalities
and lack of p21 expression has been found in human breast carcinoma and
colorectal carcinoma cells (36, 37). The relationship between p21/p53
status and 1,25 D sensitivity of human prostatic carcinoma warrants
further investigation.
Our data demonstrate that 1,25 D did not regulate the steady state
levels of p21 mRNA, nor was p21 promoter activity regulated by 1,25 D
in LNCaP cells (Fig. 8 and data not shown), suggesting that 1,25 D may
up-regulate p21 protein levels through translational or
posttranslational mechanisms. Although a functional VDRE within the
human p21 promoter has been reported in a p53-deficient cell line
cotransfected with VDR (17), we were unable to detect 1,25 D regulation
of this VDRE in LNCaP cells. The regulation of p21 protein by 1,25 D in
LNCaP cells may involve posttranslational modifications that affect
protein degradation by the ubiquitin pathway (38). It has been shown
that the ubiquitin pathway and/or phosphorylation are involved in the
regulation of CKIs such as p21 and p27 (38, 39, 40). It is possible that
1,25 D may regulate p21 protein levels in LNCaP cells by these
mechanisms or through effects on translation efficiency. The
translational or posttranslational regulation of p21 by 1,25 D may
represent a novel mechanism of action for VDR.
Taken together, our data suggest that the antiproliferative effect of 1,25 D in the highly 1,25 D-sensitive LNCaP cells is at least partially mediated by 1,25 D-mediated reduction in CDK2 activity resulting in G1/G0 cell cycle accumulation. Maximal growth inhibition of prostate cancer cells by 1,25 D is likely to require functional VDR as well as the expression and 1,25 D induction of specific proteins (including p21) that regulate progression from G1 to S phase. Thus, the work presented here provides a plausible molecular basis for the differential sensitivity of human prostate cancer cell lines to the antiproliferative effects of 1,25 D.
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Acknowledgments |
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Footnotes |
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Received July 17, 1997.
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References |
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,25-dihydroxyvitamin D3 in
seven human prostatic carcinoma cell lines. Clin Cancer Res 1:997–1003[Abstract]
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