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Departments of Pathology (C.B.J.M.C., K.S., C.C.E.M.v.E., J.A.G.M.v.d.K, J.T.) and Endocrinology and Reproduction (A.O.B.), Erasmus University DR Rotterdam 3000, The Netherlands
Address all correspondence and requests for reprints to: Dr. C. B. J. M. Cleutjens, Department of Pathology, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: cleutjens{at}pa1.fgg.eur.nl
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Introduction |
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LNCaP is an androgen-sensitive human prostatic carcinoma cell line (9) that expresses AR, but lacks GR and PR (10, 11). It was shown previously that growth of LNCaP cells, maintained in steroid-depleted culture medium, is stimulated by the addition of androgens (11). The synthetic androgen R1881, at a concentration of 10-10 M, increases the growth rate; a higher R1881 concentration is suboptimal to cell proliferation and might even have no stimulatory effect. Androgen treatment of LNCaP cells increases the messenger RNA (mRNA) level and production and secretion of prostate-specific antigen (PSA) (12, 13, 14).
In the present study we describe the generation and initial characterization of LNCaP sublines, in which the GR was stably expressed. In this way a system was established for comparison of AR and GR molecular and biological functions in a well defined cell line expressing comparable amounts of both receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmids and probes
The rat GR expression plasmid PSTC-GR(3–795) and the selection
plasmid pSV2Neo were described previously (15, 16).
PSA-61-LUC was generated by integration of a 6-kb
HindIII-HindIII (-6000/+12) fragment of the PSA
promoter in the multiple cloning site of pLUC (17). Southern and
Northern blots were hybridized with a rat (r) GR complementary DNA
(cDNA) fragment (nucleotide 2256–2543) obtained by PCR amplification
with primers: 5'-GAGTCTCACAAGACACTTCG-3' and 5'-GAAACATCCATGAGTACTG-3'
and plasmid PSTC-GR(3–795) as template using standard methods.
Northern blots were hybridized with the 320-bp
EcoRI-ClaI fragment of PSA75 cDNA (18), and a
1.2-kb PstI-PstI hamster actin cDNA fragment as a
control.
Transfections
Stable transfection. LNCaP cells were transfected according
to the calcium phosphate precipitation method essentially as described
(19), using 3 x 106 cells per 10-cm dish, 10 µg
PSTC-GR(3–795) and 2 µg pSV2Neo. After overnight
incubation with the precipitate, the culture medium was replaced by
PBS, containing 15% glycerol (incubation for 90 sec at room
temperature). Subsequently, transfected cells were incubated in culture
medium for 24 h. Next, culture medium was replaced by maintenance
medium supplemented with G418 (GIBCO BRL, Grand Island, NY) at a
concentration of 1.2 mg/ml. The resulting G418-resistant clones were
seeded into 96-well plates and selected for GR expression by
immunohistochemistry (see below).
Transient transfection. The GR-positive clones LNCaP-1B7 and LNCaP-1F5 and the parental LNCaP cells were transiently transfected according to the calcium phosphate method using 1 x 106 cells per 25-cm2 flask and 5 µg PSA-61-LUC. After overnight incubation with the precipitate, the culture medium was removed and cells were shocked in PBS, containing 15% glycerol (90 sec at room temperature). Subsequently, transfected cells were incubated in culture medium in the absence or presence of 10 nM of the synthetic glucocorticoid dexamethasone (Dex) (Sigma, St. Louis, MO) or 1 nM R1881 (DuPont NEN, Boston, MA) for 24 h. Transfections were performed three times in duplicate, using two independent plasmid isolates. Luciferase activities were corrected for variations in protein concentrations in 100-µl cell extract samples.
Luciferase assay
Cells were washed once in PBS and subsequently lysed in 300 µl
lysis buffer (25 mM Tris-phosphate, pH 7.8, 8
mM MgCl2, 1 mM dithiothreitol, 1%
Triton X-100, 15% glycerol). Next, 100 µl 0.1 µM
luciferin (Sigma)/0.25 µM ATP were added to 100 µl
extract, and luciferase activity was measured in a LUMAC 2500 M
Biocounter (LUMAC, Landgraaf, The Netherlands). After a delay of 2 sec
(according to the supplier), the light emission during 5 sec was
recorded.
Immunohistochemistry
Immunostaining for GR was performed with the monoclonal anti-GR
(rat) antibody Mab 7 (20). Cells were seeded at a density of 3 x
105 cells per well on sterile micro slides in four-well
tissue culture plates (Heraeus Instruments, Hanau, Germany) in
maintenance medium supplemented with G418 and cultured until 50–60%
confluence. Next, 10 nM Dex was added, and the incubation
was continued for 24 h. Cells were washed in PBS and fixed for 10
min in 10% phosphate-buffered formalin (pH 7.4). Subsequently, the
slides were rinsed in PBS (pH 7.4), and attached cells were made
permeable in methanol (-20 C, 5 min) and acetone (-20 C, 2 min).
After rehydration in PBS, the slides were incubated in 5% nonimmune
rabbit serum in PBS followed by overnight incubation in 1:1000 diluted
monoclonal antibody Mab7 at 4 C. Excess antibody was removed, and rGR
immunoreactivity was visualized using rabbit antimouse Ig (DAKO,
Glostrup, Denmark) as secondary antibody and mouse monoclonal
peroxidase antiperoxidase complexes (DAKO) as third-step reagent. After
three PBS washes, the slides were incubated with diaminobenzidine. The
reaction was stopped in water. Cells were counterstained with Mayers
hematoxylin.
Southern and Northern blot analysis
Total cellular DNA of LNCaP- and GR-positive sublines LNCaP-1F5
and LNCaP-1B7 was isolated using standard procedures (21). Ten
micrograms of DNA were digested with EcoRI for 16 h,
electrophoresed on 0.8% agarose gel, and transferred to a Hybond
N+ membrane (Amersham, Cardiff, UK). Filters were
hybridized at high stringency with random primed
32P-labeled probes. Both DNA transfer and filter
hybridization were carried out according to the protocol of the
manufacturer.
Isolation of total cellular RNA from the different cell lines was carried out by the guanidinium thiocyanate method (22). Glyoxal denatured RNA (10 µg/lane) was separated by electrophoresis on a 1% agarose gel and transferred to a nylon membrane (Gene Screen, DuPont NEN, Boston, MA). The blot was hybridized with random primed 32P-labeled rGR, PSA, or actin cDNA probes in 50% formamide at 42 C, using standard conditions.
Radioligand binding assay
Cells were cultured in maintenance medium until 50–60%
confluence. To deplete for steroids, cell culturing was continued in
RPMI 1640 supplemented with dextran-charcoal-stripped (dcc) serum for
48 h.
[3H]-R1881 (87 Ci/mmol) and unlabeled R1881 (methyl-trienolone) were purchased from DuPont NEN. [3H]-Dex (94 Ci/mmol) was obtained from Amersham. For radioligand-binding analysis, cells were rinsed in PBS and harvested as a single-cell suspension by trypsinization. Cells were washed four times, counted, and resuspended in ice-cold PBS. To measure the cellular AR and GR content and ligand affinity of both receptors, cells were incubated with serial [3H]R1881 dilutions (0.125–16 nM, in the absence and presence of a 100-fold molar excess of unlabeled R1881) or [3H]Dex dilutions (0.5–32 nM, in the absence and presence of a 100-fold molar excess of unlabeled Dex) for 90 min at room temperature. Free steroid was removed by extensive washing of the cells in ice-cold PBS. Radioactivity was measured in a scintillation counter. Specific binding of [3H]R1881 and [3H]Dex was calculated by subtraction of nonspecifically bound radioactivity from total bound radioactivity and used for Scatchard analysis with the radioligand-binding analysis program Ebda/Ligand by G. A. Pherson from Elsevier-BIOSOFT (Cambridge, UK). All assays were performed in triplicate.
Cell growth studies
Cells were trypsinized, seeded in RPMI medium containing 5% FCS
in 25-cm2 tissue culture flasks at a density of 5 x
104 cells, and cultured for 3 days. Subsequently, medium
was replaced by medium containing 5% dcc serum, and the incubation was
continued for an additional 3 days. At this time point (t = 0),
medium was replaced by RPMI medium containing 5% dcc serum and
indicated hormones at different concentrations. R1881 was added to
final concentrations of 10-11, 10-10, and
10-9 M; Dex was added to final concentrations
of 10-10, 10-9, and 10-8
M, respectively. Control cultures without steroids were
supplemented with 0.1% (vol/vol) ethanol. At day 4, medium was
renewed. At day 8, cells were washed in PBS and trypsinized, and the
cell number in each tissue culture flask was determined using a
Bürker’s cell-counting chamber. Experiments were performed in
triplicate.
Identification of differentially expressed genes
The mRNA Differentially Display PCR (DD-PCR) procedure used was
essentially identical to van Belzen et al. (23). In short,
LNCaP-1F5 cells were cultured in maintenance medium until 50–60%
confluence and for an additional 72 h in RPMI 1640 supplemented
with 5% dcc serum. After this period, the medium was replaced by RPMI
medium containing 5% dcc serum supplemented with 1 nM
R1881, or 10 nM Dex, or without hormone, and cells were
incubated for an additional 24 h. Isolation of total cellular RNA
from the different cell lines was carried out according to Chirgwin
et al. (22). First-strand cDNA was synthesized from 1 µg
total RNA with M-MuLV RT and a mix of three anchored oligo-T primers:
5'-TTTTTTTTTTTTG-3', 5'-TTTTTTTTTTTTC-3', and 5'-TTTTTTTTTTTTA-3'. The
resulting cDNAs from RNA of LNCaP cells grown under the three different
growth conditions were amplified for two cycles at low stringency
annealing conditions (1 min at 94 C, 5 min at 35 C, 5 min of gradual
heating from 35 C to 72 C, and 5 min at 72 C), followed by 30 cycles at
high stringency annealing (1 min at 94 C, 2 min at 50 C, and 2 min at
72 C) using a 15- to 25-nucleotide arbitrary primer. All reactions were
performed in triplicate. The resulting PCR products were separated on a
7.5% polyacrylamide gel containing 7 M urea. The dried gel
was exposed to x-ray film. Bands of interest were eluted from gel by
incubation for 1 h at 65 C in TE (10 mM Tris-HCl, pH
7.4; 1 mM EDTA). Subsequently, the eluted fragments were
amplified by an additional PCR step (30 cycles: 1 min at 94 C, 2 min at
50 C, and 2 min at 72 C). The amplified cDNAs were cloned in pGEM-T
(Promega, Madison, WI), and sequenced.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B; staining of the parental
LNCaP cells for GR was negative (Fig. 1C). Note that the morphology of
GR+ sublines is different from that of the parental cells, indicating a
thus far unexplained specific effect of the activated GR on cell
physiology. LNCaP-1B7 and LNCaP-1F5 were selected for more detailed
studies.
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;
indicated by an arrow). The rat GR probe showed
cross-hybridization to two fragments corresponding to the endogenous
human GR gene (bands a and b; see also lane 1, containing parental
LNCaP DNA). Both LNCaP-1B7 and LNCaP-1F5 DNA contained also at least
one incomplete PSTC-GR fragment (bands c and d). Densitometric scanning
of the blot revealed the presence of 6 to 7, and two complete copies of
CMV-GR cDNA in LNCaP-1B7 and LNCaP-1F5, respectively.
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). The presence of more GR mRNA in LNCaP-1F5 as compared with
LNCaP-1B7 cells, despite the higher number of integrated GR cDNA copies
in LNCaP-1B7 cells, can be due to different genomic integration sites.
Radioligand-binding assays on LNCaP, LNCaP-1B7, and LNCaP-1F5 cells
were performed to establish the number of GR and AR molecules per cell
and the respective dissociation constants for both receptors (Fig. 3).
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). LNCaP-1B7 and LNCaP-1F5
contained approximately 30,000 and 32,000 AR molecules per cell,
respectively. This 2-fold higher level of AR in the newly generated
cell lines in comparison to the parental LNCaP is probably due to a
small variation in AR content in the parental LNCaP cells. All three
cell lines showed an identical binding affinity for R1881
(Kd 1.1 nM). As expected, in the parental LNCaP cell line, binding of Dex could not be detected. From the Bmax values it could be deduced that LNCaP-1B7 contains approximately 31,000 GR copies per cell, and LNCaP-1F5 contains 115,000 copies. The Kd for Dex binding was 3.1 nM in both LNCaP sublines.
In the four lower panels of Fig. 3, the values obtained at
the two highest ligand concentrations deviate from the calculated
straight line. The apparent curvilinearity of the Scatchard data are
most likely due to the low percentage of specific binding at high
ligand concentrations, which makes these data less reliable. The
program used for Scatchard and linear regression analyses took the
differences in specific binding into account. An alternative
explanation, interaction between GR and AR, is less likely, because
experiments were done in the presence of one hormone at a time (or
R1881 or Dex).
GR activity in GR-positive LNCaP sublines
To investigate whether the GR present in LNCaP-1B7 and LNCaP-1F5
cells was functionally active, the cells were transiently transfected
with PSA-61-LUC, which contains a 6-kb PSA promoter fragment. This
promoter contains a strong androgen-dependent enhancer region,
approximately 4.2 kb upstream of the transcription start site of the
PSA gene (17, 24). As expected, PSA-61-LUC showed clear, R1881-induced
luciferase activity in transfected parental LNCaP cells. No luciferase
activity was detected upon incubation of the PSA-61-LUC-transfected
parental LNCaP cells with Dex (Fig. 4).
Transient transfection of LNCaP-1B7 and LNCaP-1F5 cells with the
PSA-61-LUC construct resulted in a comparable R1881-induced luciferase
activity, both in absolute luciferase activity and in induction level
(1310- and 1940-fold, respectively). Dex induced a slightly higher
PSA-61-LUC activity, clearly indicating the transactivating capacity of
the GR encoded by the stably integrated rGR expression vector (Fig. 4).
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). In the parental LNCaP cell line, PSA
mRNA expression was induced by R1881 but not by Dex. In contrast, in
the two GR-positive clones, Dex treatment clearly resulted in
stimulation of PSA mRNA expression, although to a slightly lower level
(approximately 3-fold) than R1881 up-regulated PSA mRNA.
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). Similar negative results were
obtained with hydrocortisone (data not shown). These results clearly
indicated that GR was unable to replace AR in growth stimulation of
LNCaP cells.
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), and not
from RNA of Dex- incubated LNCaP-1F5 cells, or cells incubated in the
absence of hormone. Using fragment 21.1 as a hybridization probe, on a
Northern blot of LNCaP-1F5 RNA, a mRNA of approximately 5 kb was
detected, and the differential expression pattern was confirmed (Fig. 7B). The sequence of the 504-bp 21.1 fragment is available in GenBank
under accession number AF007835.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At least part of the effects of steroid hormone receptors on gene expression is on transcription initiation. Upon ligand binding, steroid receptors interact with specific DNA sequences (HREs) and regulate the transcription of target genes. The GGT/AACAnnnTGTTCT consensus sequence for high-affinity DNA binding of GR, MR, PR, and AR is identical (5, 6, 7, 8). Despite this common DNA-binding site, the different receptors mediate distinct cellular responses. Many independent mechanisms to achieve specificity of the steroid hormone response have been proposed (see for reviews Refs. 3 and 25). These include differential affinities to natural receptor-binding sites, or to binding sites in their natural DNA context (26, 27, 28, 29, 30), differential affinity to general and specific transcription factors (31, 32, 33, 34), differences in interaction with receptor-specific accessory proteins or coactivators (35, 36, 37), differential modification of specific chromatin structures (38, 39), differences in cellular concentration of the specific receptors (40), and variations in ligand availability (41, 42).
The observation that PSA mRNA was Dex inducible in the GR-expressing LNCaP sublines showed that the steroid receptor content determines, at least in part, the specific activation of the PSA gene in wild-type LNCaP cells. Transient transfection of the 6-kb PSA promoter to the LNCaP sublines resulted also in activation by both AR and GR. In contrast, in the case of the MMTV promoter, differences have been reported on the effect of GR and PR on transiently transfected reporter gene constructs and stably integrated plasmids (38, 39). It appears that the native chromatin structure can prevent PR activation and permits GR stimulation of the stably integrated target gene.
AR activation of the PSA promoter involves at least three androgen response elements, two in the 600-bp proximal promoter region and one in a far upstream enhancer region (17, 43). Although ultimate PSA promoter induction is comparable for AR and GR, it cannot be excluded as yet, that AR and GR affect the individual regulatory regions differentially. Such an observation has been made for GR and PR activation of the MMTV promoter, which contains four HREs. Differences might be accounted for by distinct chromatin structures over the individual HREs, and the ability of the different receptors to affect these structures, and/or the interaction with other specific transcription factors (34).
As shown above, in contrast to normal human prostate tissue, GR expression cannot be detected in wild type LNCaP cells (44). However, in normal prostate highest GR expression is in the stromal compartment. In epithelial cells expression of AR appears much higher than GR expression. In prostate cancer, GR expression seems even to decrease. Therefore, GR might not be a major factor in PSA expression. No data are available relating to PR expression in prostate tissue, another candidate for regulation of PSA expression (17).
The stimulatory effect of androgens on LNCaP cell proliferation shows a bell-shaped dose-response curve (11, 45). At low androgen concentrations (up to 10-10 M R1881), LNCaP cells proliferate in a dose-dependent manner; at higher hormone concentration, the proliferation rate is less. The molecular mechanism of growth stimulation by androgens, including the remarkable dose response, is not fully understood, although it has been proposed that TGFß1 mediates at least part of the growth arrest observed at high androgen concentration (46). The cell growth studies clearly demonstrated that Dex was unable to induce proliferation of LNCaP-1B7 and LNCaP-1F5 cells. The molecular mechanism responsible for the differential effects of glucocorticoids and androgens on growth remains to be investigated. In a probably oversimplified view it can be hypothesized that a limited number of differentially expressed genes are involved. Differential AR and GR regulated TGFß and other growth factors or inhibitors, and their corresponding receptors should be considered in this regard.
The novel clone 21.1 described here might be another candidate involved in a specific AR function in LNCaP. Isolation and characterization of full-length 21.1 cDNA and the accompanying gene, which might provide information on 21.1 function and regulation of expression, are in progress.
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Acknowledgments |
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Footnotes |
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Received March 20, 1997.
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