Division of Biochemistry (A.D., F.C., P.A., W.R., B.P.) and Laboratory for Experimental Medicine and Endocrinology (J.W., W.H.) Faculty of Medicine University of Leuven B-3000 Leuven, Belgium
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ABSTRACT |
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INTRODUCTION |
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The cystatin-related protein genes (crps) are interesting candidates to study the mechanisms by which androgen-dependent gene expression and tissue specificity are obtained. CRPs are abundant glycoproteins almost exclusively synthesized in ventral prostate and lacrimal gland of the male rat (7). They are encoded by a multigene family (8) of which we have isolated two 90% homologous genes, crp1 and crp2 (9). A 20-kDa protein gene, identical to crp1, has been described by Ho et al. (5). In spite of the high homology between crp1 and crp2, their transcription is known to be differentially influenced by androgens in vivo. The crp1 response to androgens is fast and crp1 mRNA is detected in both ventral prostate and lacrimal gland. It can also be induced in female rats by androgen administration (10). The crp2 mRNA is exclusively detected in the ventral prostate, and its transcription is less sensitive to androgens (7).
The following report describes the search for functional AREs in these androgen-regulated crp genes. Up till now, these regulatory elements have mainly been found in the promoter regions for androgen-controlled genes such as those encoding prostate-specific antigen (PSA), sex-limited protein (Slp), probasin (PB), and mouse vas deferens protein (MVDP) (11, 12, 13, 14, 15). However, functional AREs have been detected in the first intron of the C3(1)-gene (16, 17) and the ninth intron of the ß-glucuronidase (GUS)-gene (18).
Chimeric constructs, containing fragments of a large region encompassing the 5'-upstream and intron 1 sequences of both crp genes, have been examined in the androgen-sensitive human breast cancer cell line T-47D. While fragments from intron 1 confer a weak to moderate androgen responsiveness to a reporter gene after transient transfection of chimeric constructs in these cells, a more significant response was observed with fragments originating from the transcription start point-containing region. A detailed analysis of these latter fragments has been made.
The deoxyribonuclease I (DNaseI) footprinting technique was used to locate AR-binding sites using a recombinant androgen receptor DNA-binding domain (AR-DBD) (19). A gene-specific footprint was detected in the coding sequence of the first exon of crp2 and a common footprint for crp1 and crp2 in their 5'-upstream regions. The protected sequences themselves were tested by gel retardation for AR-DNA complex formation and by mutational analysis to determine their role in the androgen-dependent response.
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RESULTS |
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As this transcriptional inactivity could be the result of cell
type-specific promoter functioning or the presence of a silencer in the
crp gene, a different approach was followed. The same
promoter fragments, but also several subfragments generated from this
region (see Materials and Methods), were tested in chimeric
constructs, containing the TK-CAT reporter gene. The TK-promoter can
activate gene transcription in T-47D and LNCaP cell lines (13, 16, 20).
Under these conditions, the chimeric constructs can still be tested for
the property of the inserted fragment to confer androgen responsiveness
to the heterologous promoter. Transient transfections in T-47D cells
revealed that a consistent 2- to 3-fold stimulated CAT activity can be
obtained in the presence of 10-8 M 5DHT or
10-9 M R1881 but only with vectors containing
the crp1 Fragment (crp1F; nt -207 to +89) and
crp2 Fragment (crp2F; nt -273 to +88) (Fig. 2
).
Several constructs, containing other regions of the 5'-upstream
sequences (for crp1 nt -584 to +11 and crp2 nt
-1392 to +46 see Fig. 2
; crp1 promoter fragment nt -584 to
-207 and crp2 promoter fragments nt -1037 to -586 and nt
-585 to -273, results not shown), did not induce an
androgen-dependent transcription.
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We reexamined all chimeric constructs in T-47D cells after
cotransfection with pSVAR0, an expression plasmid for the human AR.
These conditions explicitely demonstrate the involvement of the AR in
the androgen-dependent transcription of the crp constructs.
Essentially the same fragments are responsive to androgens but,
depending on the constructs tested, their hormone-dependent
transcription is slightly to highly increased (Fig. 2). While almost
without effect on the control constructs, the level of androgen-induced
CAT-synthesis for the crp2F (-273/+88) construct increased
substantially from 3.1- to 13.6-fold. Such an increase was not observed
for the crp1F (-207/+89) construct (Fig. 2
). Both steroid
hormones, 10-8 M 5
DHT and 10-9
M R1881, produce identical results.
The androgen-dependent transcription of some constructs containing
large intron 1-derived fragments (nt 22742857 for crp1, nt
19552897 for crp2) is also increased after cotransfection
of an AR expression vector (Fig. 2). Further analysis showed that the
androgen responsiveness could not be traced back to a single small
segment as both the D1 (2634/2857 for crp1, 2654/2897 for
crp2) and D2 (2503/2633 for crp1 and 2525/2653
for crp2) subfragments displayed a moderate 2-fold induction
by the hormone.
To find out whether the steroid response of the constructs containing
the crpFs or intron 1 fragments is androgen specific, we
tested the effect of a series of steroids in T-47D cells without SR
cotransfection. The androgens 5-DHT (10-5 to
10-10 M), testosterone (10-6 and
10-7 M), and R1881 (10-9
M) are all able to induce a 2- to 3-fold induction of the
crpF-TK-CAT constructs. A similar level of induction is
observed for the synthetic progestin R5020 (10-9
M) and for dexamethasone (10-6 M).
The crp1F (-207/+89)-TK-CAT and crp2F
(-273/+88)-TK-CAT constructs, cotransfected with either an expression
plasmid for the human glucocorticoid receptor (hGR) or human androgen
receptor (hAR) also revealed similar steroid hormone inducibility in
COS1 cells treated with the corresponding hormone. CAT transcription,
mediated by the different intron 1-containing constructs (Fig. 2
), can
also be efficiently stimulated by adding 10-6
M dexamethasone to the T-47D cells cotransfected with
pRSVhGR (results not shown). The induction factors are similar to those
observed for the same constructs, cotransfected with pSVAR0 in T-47D
cells and treated with 10-9 M R1881.
Localization of AR-DBD Binding Sites by DNaseI Footprint
Analysis
As the small crp2F (-273/+88) fragment confers a
strong and consistent androgen inducibility to the CAT gene, we have
focused on this fragment and the corresponding region of the
crp1 gene. DNaseI footprinting was performed on both
homologous fragments, using the purified recombinant AR-DBD fusion
protein (19).
In crp2F (-273/+88) an AR-binding site
ARBSd/crp2, displaying an ARE-like motif, is detected
downstream of the transcription start point (nt +40 to +63) (Fig. 3A). The start codon of the CRP2 protein is included in
this ARE-like sequence. The size of the protected region (24 nt)
furthermore indicates that the AR-DBD molecules interact as dimers with
the DNA. A clear hypersensitive site appears at the upstream boundary
of the protected region (Fig. 3A
, lane 2) In the corresponding
crp1 sequence, which displays one A to G base exchange and
the insertion of an A, no footprint was detected (Fig. 3B
, lane 2).
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A second, but fainter AR-binding site ARBSu/crp2 (nt -136
to -115) was observed in the 5'-upstream region on crp2F
(-273/+88) (Fig. 3C, lane 2). The corresponding region in
crp1F (-207/+89) was also protected by AR-DBD against
DNaseI digestion, resulting in a footprint ARBSu/crp1 (nt
-142 to -121) (Fig. 3D
, lane 2). These footprints are located at
identical positions in both genes as can be deduced from the sequence
alignment for optimal sequence identity by the PC Gene program (9). The
protected sequences mutually differ in two nucleotides. Protein A, to
which the AR-DBD is linked, is not responsible for the footprint as it
does not confer protection of the ARBSu/crp1 against DNaseI
(Fig. 3D
, lane 4). Here too, mutation of the 3'-half-site 5'-TGTACT-3'
sequence of the putative AREs to a PvuII restriction site
abolishes protection, even when the AR-DBD is added at 5-fold higher
concentration (results not shown).
Full-Length AR Binds Differentially to the crp-ARBS
Sequences
To compare the affinity of the AR for the binding motifs observed
in footprint analysis, gel retardation experiments were performed with
labeled ds oligonucleotides encompassing the complete protected
sequences. For this analysis, a full-size AR overexpressed in HeLa
cells by the vaccinia system was used instead of the AR-DBD recombinant
protein. Although the latter protein produces a stable protein-DNA
complex with the core II motif taken from the C3(1) gene intron, no
complexes were retarded using oligonucleotides containing the
ARBS-derived sequences (6). The full-length AR interacts with core II
(Fig. 4A, lane 36) but not with its mutated form, even
when AR-Ab are added (Fig. 4A
, lane 89) because only weak bands of
nonspecific complexes also present in HeLa nuclear extract (Fig. 4A
, lane 1) are seen. However, with the full-length AR, not only complex
formation was observed with the core II sequence, but also with the
oligonucleotides containing ARBSd/crp2 and even with the
ARBSu/crp2 and ARBSu/crp1 sequences. Using an
equal labeling specificity of the different probes, the detection of
the DNA/AR complex requires an almost 6-fold longer exposure time (Fig. 4B
). The complex formation could be improved by the addition of AR
antibodies, especially with the ds probes containing sequence motifs of
lower receptor binding affinity, and resulted in a supershift of the
specific AR-DNA complex. Furthermore, competition experiments revealed
the specificity of these interactions. The AR-ARBSd/crp2
complex was absent after addition of a 200-fold excess of the unlabeled
ds oligonucleotide (Fig. 4B
, lane 8). Although at this competitor
concentration, no effect was seen on the amount of AR-core II complex
formed (Fig. 4B
, lane 3), this competition can be observed when the
film is not overexposed (Fig. 4A
, lane 4). This indicates that the
receptor has a lower affinity for this competitor sequence than for the
core II motif, which displays the highest homology with the consensus
GRE/ARE.
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Finally, we could demonstrate that ARBSd/crp2 is an enhancer
sequence. Indeed, inserted in the opposite orientation
crp2(F-) (+88/-273) in front of the promoter, thereby
increasing the distance between ARBSd/crp2 and other
regulatory elements in the TK-promoter from 38 bp to 220 bp, it is
still able to induce a moderate androgen-dependent gene transcription
(Fig. 5).
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DISCUSSION |
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As no CRP-synthesizing epithelial cell lines are available, T-47D cells have been used. They contain a functional AR, and androgen responsiveness of several fragments from other steroid-controlled genes has been successfully investigated in this cell line (13, 16, 21). To take into account the possibility of cell type-specific gene expression (22), the hormone-dependent transcription of some chimeric constructs has also been tested in LNCaP and COS1 cells.
Putative ARE-like sequences were detected in the 5'-upstream regions of the crp genes (9), but identification of these elements by their functional analysis in the context of the endogeneous crp promoter has been unsuccessful. Similar observations of promoter inactivity have been made for constructs of the 20-kDa protein gene transfected into CV1 cells (5) and of the C3(1) gene when tested in T-47D and CV1 cells (16, 17).
Using the viral TK-promoter, which is functional in a wide range of
different cell lines, a low but consistent androgen-induced
transcription of CAT was seen in transiently transfected T-47D cells
for the crpF-TK-CAT (-207/+89 for crp1 and
-273/+88 for crp2) constructs and those containing intron
1-derived fragments (Fig. 2). Cotransfection of pSVAR0 in T-47D cells
did improve the androgen-dependent transcription of some of these
constructs. Unexpectedly, the induction level of the crpF
containing TK-constructs depends on the batches of T-47D cells taken
for transfection.
Three successive samples of T-47D cells, obtained from ATCC (Rockville, MD), display a clear R1881-dependent transcription of crp2F (-273/+88)-TK-CAT. However, the highest transcriptional enhancement by R1881 (17-fold induction) has been observed with the crp1F(-207/+89)-TK-CAT construct. This induction level has been consistently seen in a large number of transfection experiments but only in one specific, fast proliferating T-47D cell batch, which displays an insulin-independent growth.
Our transfection results of crp1 and the observations made by Ho et al. (5) revealed a cell-specific androgen response. In T-47D cells, we did not find any androgen responsiveness for the 5'-upstream gene fragment (-584/+11) whereas these authors have detected a 2-fold induction of CAT transcription by androgens when a fragment (-582/-182) from this region was tested in CV1 cells. This discrepancy could be due to the presence of cell type-specific silencer elements that affect the androgen-dependent transcription as has been shown for some other genes (12, 13, 14).
For constructs containing intron 1 fragments and tested in T-47D cells, the level of androgen inducibility is always lower than the one observed with the crpF constructs. In contrast, similar intron 1 fragments reveal a higher androgen responsiveness in comparison with the constructs containing the promoter region when examined in CV1 cells (5). In addition, the hormone selectivity of the intron fragments observed by Ho et al. (5) in CV1 cells has not been seen in T-47D cells. This lack of hormone-specific response has also been observed during promoter studies of the MVDP gene in the T-47D cell line (15) and of the PSA gene in the homologous cell line LNCaP (20).
DNaseI footprinting on the crpF fragments with a recombinant fusion protein containing the AR-DBD (19) reveals two windows in crp2F (-273/+88) and one in crp1F (-207/+89). The region shielded from DNaseI attack indicates that the AR-DBD interacts as a dimer, as its size is comparable to the one previously observed on the core II motif in the intron 1 fragment of the C3(1) gene and on the distal HRE of the long terminal repeat region in MMTV (23).
All three AR-binding motifs differ at position -2 of the highly conserved consensus sequence for an ARE (5'-AGAACAnnnTGTTCT-3') (2) as deduced from functional elements identified in a number of androgen-regulated genes (15). This base exchange strongly affects the stability of the DNA-receptor complex (24, 25). This has also been demonstrated in footprinting analysis on the crpFs by competition experiments with either the core II motif of C3(1) or the ARBS/crp derived probes. Moreover, mobility shift analyses using the AR-DBD only reveal DNA-protein complex formation with the core II ds oligonucleotides, and not with the sequences containing ARBS/crp motifs. However, the full-size AR interacts both with the core II- and ARBSd/crp2-sequences. ARBSu/crp1- and ARBSu/crp2-receptor complexes could only be clearly detected after stabilization with AR-antibodies, indicating AR interaction with gene fragments displaying weak affinity binding sites (5, 14).
The ARBSd/crp2 contains an ARE-like motif that very well resembles the proximal ARE (5'-TGAAGTtctTGTTCT-3') described in the MVDP gene promoter (15). The corresponding region in the crp1 gene shows no AR-DBD interaction. This is most probably due to the insertion of an A in the spacer region between the two 6-bp half-sites of the binding motif. The 4-bp spacing completely destroys binding of SRs to their recognition site as has been clearly demonstrated for the GR and PR (6, 24, 25).
The role of these AR-binding sites, ARBSd/crp2, ARBSu/crp2, and ARBSu/crp1 showing a moderate to weak affinity for the receptor, has been identified in the crpFs. ARBSd/crp2 consistently confers androgen response to the heterologous promoter in T-47D cells cotransfected with an hAR expression plasmid and supplemented with R1881. The location of the ARBSd/crp2 in the coding region of exon 1 is most unusual, as AREs identified downstream of the transcription start point have thus far been found in introns (16, 18). Its function as enhancer has been confirmed by the fact that crp2(F-) (+88/-273)-TK-CAT is still able to confer androgen-dependent induction. The decrease in induction level is most probably the result of a positional effect. Increasing the intervening sequence between a HRE and other regulatory elements displaying cooperative interaction through their bound transcription factors can indeed affect the steroid-dependent transcription of a reporter gene (26, 27, 28). The mutation of the 3'-half-site of ARBSd/crp2, which does not change the overall configuration of crp2F (-273/+88)-TK-CAT, completely abolishes its androgen-dependent transcription.
The role as an ARE of the common ARBSu/crp motifs is less
clear. The ARBSu/crp2 does not seem to participate in
controlling the androgen response of the crp2F (-273/+88)
chimeric construct in the T-47D clones that we are currently using.
Mutation of ARBSu/crp2 hardly affects the androgen-dependent
transcription of the mutated construct (Fig. 5). These results are
substantiated by the observation that another construct containing only
the 3' half of crp2F (-98/+88), and thus only the
ARBSd/crp2 motif, displays almost the same level of
induction as the complete fragment. Nevertheless, in a series of
experiments using a fast proliferating, insulin-independent T-47D
clone, we observed that destroying the AR binding to
ARBSu/crp2 had an effect on the androgen responsiveness. The
crp2F/ARBSum construct showed a significant drop
(40%) in steroid-dependent transcription (results not shown). These
results could indicate that a functional interaction may exist between
the two ARE-like elements. Similar observations of cooperative
interaction between AREs have been described for the probasin gene
promoter (14). A further proof that AR recognition motifs displaying
weak affinity for the receptor can still function as strong AREs in
transfection experiments, when positioned in their appropriate DNA
context and transfected in a responsive T-47D clone, is demonstrated
for the crp1F (-207/+89) construct. This fragment,
containing a single AR-binding site (ARBSu/crp1) with a
sequence and receptor binding property almost identical to the
ARBSu/crp2 motif, can confer a 17-fold induced transcription
by R1881. The fact that a small mutation in the 3'-half-site of the
ARBSu/crp1 sequence, destroying the AR binding, can
completely shut off the androgen-dependent transcription of the
reporter gene clearly underlines its function as an ARE. In
vitro binding experiments reveal that the ARBSu/crp1
regulatory element is part of a complex androgen response unit. DNaseI
footprinting on the crp1F (-207/+89), using nuclear
extracts of rat ventral prostate and lacrimal gland tissue, has shown
that an array of binding sites for different transcription factors is
flanking this ARBSu/crp1 site and the pattern of footprints
produced is distinct for the crp1 and crp2
promoter (unpublished results).
The fact that the highly homologous segments crp1F and crp2F confer a quite different hormone responsiveness when tested as chimeric constructs in transient transfections may provide some potential clues to understanding the differential response of the two genes as seen in vivo. Moreover, functional AREs have been identified in these androgen-responsive crp promoter fragments. They differ in number and affinity as demonstrated by in vitro binding studies, and the sequences are markedly different from the consensus ARE. Furthermore, the location of the ARBSd/crp2 motif is of particular interest. Indeed, while most AREs detected up till now are found in the 5'-upstream and intron regions, the ARBSd/crp2 is located in exon 1 over the translation start site of CRP2, and therefore it is the first ARE described in a coding sequence of a gene.
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MATERIALS AND METHODS |
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Construction of CAT Fusion Plasmids
The crp-gene subfragments were obtained either by
restriction enzyme cleavage or PCR amplification and were cloned into
the pBLCAT2 vector, containing a TK promoter linked to a CAT reporter
gene (30). pBLCAT2 was digested with SalI and used as such
or made blunt-ended by a fill-in reaction. Recombinant clones with
specific inserts were selected, and vector/insert overlapping regions
were analyzed by sequencing.
A BamHI-ScaI genomic crp1 fragment (nt -584 to +292) (9), containing the promoter region, was digested by HaeIII, and the purified subfragments of 379 bp and 296 bp were cloned (crp1PF1, -584/-207 and crp1F, -207/+89). For the crp2-promoter region, an AccI-ScaI fragment (nt -1037 to +291) was treated with HaeIII and fragments of 454 bp, 312 bp, and 358 bp were inserted in the vector (crp2PF1, -1037/-586; crp2PF2, -585/-273; crp2F -273/+88). From the 358-bp fragment that contains the transcription start point, two subfragments were generated by HinfI-digestion and subcloned (crp2Fu -273/-98 and crp2Fd -97/+88).
The crp1-promoter fragment (-584/+11) was obtained by serial deletion at the 3'-end using the Erase-a-base system (Promega, Madison, WI). Religated plasmids were transfected in XL1-Blue cells (Stratagene, La Jolla, CA), and clones containing the recombinant plasmids, displaying inserts of about 600 bp, were analyzed by sequencing to determine the exact 3'-end of the gene insert. After SalI/NsiI digestion, the promoter fragments were cloned in the SalI/PstI double-digested vectors pCATEnh vector (Promega) or pBLCAT2 (crp1P: -584/+11).
A fragment containing the complete 5'-upstream region of the crp2-gene (nt -1392 to +46), cloned in pGEM-7Zf(+), has been made by PCR (Gene Amp Reagent kit with amplitaq; Cetus Inc, Berkeley, CA) with the primers 5'-aaagtcgacagatctATCCAGAGTCCTGGG-3' (nt -1392/-1375) and 5'-aaagtcgacggatccTCAGATATGAAAGGG-3' (nt +30/+46). These primers were designed to contain restriction sites at their 5'-ends. All PCR reactions have been performed on plasmid DNA, prepared either by CsCl gradients or Qiagen columns. PCR reactions were carried out according to the protocols. After SalI digestion, the promoter fragments were cloned in the SalI-site of the pCATEnh vector (Promega) or pBLCAT2 (crp2P: -1392/+46).
Genomic fragments of intron 1 were derived from the 3445-bp crp1 BamHI fragment (nt -574 to +2857). After digestion with HinfI, a segment of 474 bp (nt 22742857) was selected. D1 and D2 fragments were obtained from HindIII digestion of the same BamHI crp1 fragment, followed by a DdeI digestion. The fragments of interest are crp1D1 (nt 26342857) and crp1D2 (nt 25032633). For crp2, a BamHI fragment (nt -1393 to +2897) was cleaved with Sau3AI yielding a fragment of 944 bp (nt 29552897). crp2D1 and crp2D2 were derived from the same 4287-bp BamHI fragment, digested by XbaI and subsequently digested by DdeI. The fragments crp2D1 (nt 26542897) and crp2D2 (nt 25252653) were subcloned in the pBLCAT2 SalI site, which had been made blunt-ended.
PCR-mediated mutagenesis was performed according to Higuchi et al. (31). For crp1, the oligonucleotides 5'-TGGGTGAGGGAACAAGCAGCTGATAGGGAATGAAGTT-3' (-145/-210) and 5'-AACTTCAATCACTATCAGCTGCTTGTTCCCTCACCCA-3' were used for the generation of the ARBSu/crp1 mutant. On crp2F, oligonucleotides 5'-TGGGTGAGGTAAAAAGCAGCTGATAGGGAATGAAGTT-3' (-239/-204) and 5'-AACTTCATTCCCTATCAGCTGCTTTTTACCTCACCCA-3' were taken to obtain the ARBSu/crp2 mutant and a combination of the oligonucleotides 5'-TATCTGAGAAGAAAACAGCTGAAACCCTATGTGGCA-3' (+37/+73) and 5'-TGCCACATAGGGTTTCAGCTGTTTTCTTCTCAGATA-3' results in the ARBSd/crp2 mutation. The PCR products were subcloned in a pCRScript vector (pCR-Script SK(+) Cloning kit, Stratagene). Inserts were tested for the presence of the required mutation by restriction analysis with PvuII before subcloning in pBLCAT2. For crp1, the clone carrying the mutation in ARBSu/crp1 (crp1F/ARBSum) was obtained; for crp2, crp2F/ARBSum carries the mutation in ARBSu/crp2; crp2F/ARBSdm has the mutation in ARBSd/crp2. All inserts produced by PCR were verified by complete sequencing of the inserts.
Cell Culture and Hormones
Three batches of the human breast cancer cell line T-47D were
ordered at different occasions at the American Type Culture Collection
(Rockville, MD). Batch 1 contained ±125 fmol AR, ±50 fmol GR, and
±480 fmol PR per mg protein. This company also provided the human
lymph node carcinoma of prostate cell line LNCaP and the African green
monkey kidney cell line COS1. T-47D cells were grown in DMEM containing
1000 mg/liter glucose, supplemented with 100 U/ml penicillin, 0.1 mg/ml
streptomycin, 4 µg/ml insulin, and 10% FCS (Sera-Lab, Sussex,
England; or GIBCO-BRL, Gaithersburg, MD). LNCaP cells were grown in the
same medium, supplemented with L-glutamine (2
mM). For COS1 cells, DMEM containing 4500 mg/liter glucose,
20 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin,
and 5% FCS was used as growth medium. All cell lines were regularly
checked for Mycoplasma contamination (Mycoplasma detection
kit, Boehringer Mannheim, Mannheim, Germany).
T-47D and COS1 cells were transfected by the calcium phosphate coprecipitation transfection method (16). For cotransfections, 4.5 µg plasmid DNA were used to which 0.25 µg pCMV-LUC control vector and 0.25 µg pSVAR0 were added. The transfection method by lipofectin has been optimized for LNCaP cells, according to the accompanying protocols. pMMTV-CAT has been described by Cato et al. (32) and was used as a positive control on hormone response for each set of transfection experiments. To allow comparison of the transcription conferred by each of the promoters, the CAT activity was corrected for differences in transfection efficiency, as measured by the luciferase activity resulting from the cotransfected pCMV-LUC-vector.
The cells were maintained in the presence or absence of
10-5 to 10-10 M
5-dihydrotestosterone (5
-DHT), 10-6 or
10-7 M testosterone, 10-9
M Methyltrienolone (R1881), 10-6 M
dexamethasone, or 10-9 M promegestone (R5020).
Culture medium with or without hormone was replaced every 24 h.
Transfections were repeated at least three times, using at least two
different plasmid preparations.
CAT and Luciferase Assays
Cells were harvested in 500 µl of 1x reporter lysis buffer
(Luciferase cell culture 5x lysis buffer, Promega) and allowed to
stand at room temperature for 15 min. Lysed cells were collected,
transferred to Eppendorf tubes, vortexed for 1 min, placed on ice for
10 min, and again vortexed for 1 min. After the lysate was spun for 3
min at 13,000 rpm, supernatant was transferred to a new tube. Protein
concentration of supernatant was determined with the Bradford reagent
in microtiter plates (DC Protein Assay System, Bio-Rad, Richmond, CA).
For corresponding samples with and without hormone, protein
concentration was equalized. Luciferase activity was measured on 10
µl of lysate in a MicroLumat LB96P (EG&G Berthold, Badwildbad,
Germany) using the luciferase assay kit (Promega) according to the
protocol provided by the company.
Lysate (300 µl) was used to perform the CAT assay with acetyl coenzyme A (AcCoA) obtained from Pharmacia (Piscataway, Uppsala, Sweden) and [14C]chloramphenicol (CAM) from Amersham (Little Chalfont, England). The labeled CAM and derivates were extracted with ethylacetate (Janssen Chimica, Beerse, Belgium), spotted on TLC plates, and separated in CHCl3-MeOH (19:1). Radioactive spots were quantified by use of the PhosphorImager and the accompanying software (Molecular Dynamics, Sunnyvale, CA). Relative induction factors are expressed as the mean and SEM of at least three independent experiments.
Labeling DNaseI Footprint Fragments
The restriction fragments of crp1 (nt -207 to +89,
footprint fragment crp1F) and of crp2 (nt -273
to +88, footprint fragment crp2F), subcloned in the
EcoRI site of pGEM-7Zf(+), were used to prepare the
end-labeled probes necessary in DNaseI footprinting.
For the upper strand, pGEM-7Zf(+) plasmids, containing the
crp1F or crp2F, were digested with
ClaI, labeled with Klenow DNA polymerase (Pharmacia) and
[-32P]dCTP (Amersham International) and redigested
with SphI. For the lower strand, the first digestion was
with XbaI, the ends filled in with
[
-32P]dCTP, and the fragments were subsequently
digested with SacI. The labeled DNA fragments were purified
on a 4% nondenaturing polyacrylamide gel (acrylamide-bisacrylamide
29:1; 1x Tris-borate-EDTA) by electrophoresis for 2 h at 120 V.
The bands were cut out of the gel after they had been localized by
means of an x-ray film, and DNA was extracted from the gel fragments by
electro-elution. DNA was precipitated with ethanol and redissolved at
10,000 cpm/µl in water.
DNaseI Footprinting Analysis
DNaseI footprinting experiments were performed according to
Celis et al. (29). An almost pure fusion protein of the DBD
(amino acids 540607) of the AR, linked to protein A and expressed in
Escherichia coli, was obtained by affinity chromatography on
IgG Sepharose (19).
Labeled probe (30,000 cpm) was incubated with 600 fmol to 5 pmol AR-DBD fusion protein for 20 min at 20 C. For the competition experiments, the competitor DNA was also added at this stage. Digestion with 100500 mU DNaseI (Boehringer Mannheim, Mannheim, Germany) was performed during 60 sec at 20 C in a final volume of 50 µl. The digested DNA probe was analyzed on a denaturing (7 M urea) 8% polyacrylamide sequencing gel.
The G and (A+C)-degradation reactions were performed, according to Maxam and Gilbert (33). For each footprinting experiment, the DNaseI-treated end-labeled probes are aligned with the sequence produced by this partially chemically cleaved probe to locate putative sites of interaction.
Oligonucleotides
Synthetic complementary oligonucleotides were used as ds
probes that were labeled for electrophoretic mobility gel shift assay
(EMSA). Unlabeled oligonucleotides were taken as competitor in the
DNaseI footprinting and EMSA experiments. The core II containing
oligonucleotide (5'-agcttACATAGTACGTGATGTTCTCAAGgtcga-3') has the ARE
motif of the C3(1) intronic fragment. Its mutated counterpart carries a
mutation in the 3'-half-site of this ARE motif
(5'-agcttACATAGTACGTGATTTTCTCAAGgtcga-3'). NF1-Ad
(5'-ATTTTGGCTACAAGCCAATATGAT-3') contains the binding site in
adenovirus for nuclear factor 1. ARBSu/crp1
(5'-agcttGTGAGGGAACAAGTGTACTATAGgtcga-3'), ARBSu/crp2
(5'-agcttGTGAGGTAAAAAGTGTACTATAGgtcga-3'), and ARBSd/crp2
(5'-agcttTCTGAGAAGAAAATGTACAAAACCatcga-3') represent the sequence
motifs protected by AR-DBD in DNaseI footprinting on the crppromoter fragments.
EMSA
EMSA was performed as described by De Vos et al.
(34). The full-length AR has been prepared in a vaccinia system (35).
[-32P]dATP-labeled probe (20,000 cpm) was incubated
for 20 min at 20 C in a final volume of 20 µl with 2.55 µg HeLa
cell nuclear extract, either containing the AR (510 fmol AR) or not.
The samples were separated on a 4% polyacrylamide gel
(acrylamide-bisacrylamide, 29:1) in 0.25 x TBE-buffer. To some of
the reaction mixtures, rabbit polyclonal antibody (Ab) against the
N-terminal domain of the AR (AR-Ab) have been added. For the
competition experiments in gel retardations, 100- or 200-fold excess of
unlabeled probe was added to the incubation mixture.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported in part by Grant Geconcerteerde Onderzoeksactie van de Vlaamse Gemeenschap and by Grant 3.0048.94 from the Belgian Fonds voor Geneeskundig Wetenschappelijk Onderzoek. A.P. is supported by a scholarship of the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie. C.F. is Senior assistant of the Nationaal Fonds voor Wetenschappelijk Onderzoek.
Received for publication September 23, 1996. Revision received April 14, 1997. Accepted for publication April 17, 1997.
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REFERENCES |
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