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The Albert Einstein Comprehensive Cancer Center (A.T.R., M.F.,
C.W., C.A., R.G.P.) Division of Hormone-Dependent Tumor Biology
Department of Developmental and Molecular Biology Albert Einstein
College of Medicine Bronx, New York 10461
Department of
Internal Medicine (M.J.M.) University of Texas Southwestern Medical
Center, Dallas Dallas, Texas 75235-8857
Department of
Surgery and Genetics (Z.S.) Stanford University School of
Medicine Stanford, California 943054
Hematology-Oncology
Division (S.P.B.) Department of Medicine Beth Israel
Hospital Boston, Massachusetts 02215
Department of
Physiology (O.A.J., J.J.P.) Institute of Biomedicine and Department
of Clinical Chemistry University of Helsinki FIN-00014
Helsinki, Finland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Coactivator proteins for the steroid receptors can be classified into structurally related groups and include the cointegrators [CREB-binding protein (CBP) and the related functional homolog p300 (8)], the steroid receptor coactivators (SRC-1), (also referred to as p160/NCoA-1 or ERAP-160) and related family members (9), and the steroid receptor RNA activator (SRA) (10). SRC family proteins and several other nuclear receptor coregulators contain a conserved leucine-rich domain (LXXLL) and an adjacent basic region that together serve as a nuclear receptor interaction motif (9, 11). Coactivator recruitment to a nuclear receptor involves sequential docking through the basic region to charged residues on the receptor, and then docking of the LXXLL motif (11). The cointegrator proteins, p300/CBP and the SRC family, share the capacity to acetylate histones, which often correlates in part with their transcriptional coactivator function (12). The enhancement of transcriptional activity by p300/CBP requires a bridging function to associate transcription factors with the basal transcription apparatus (13), and both intrinsic and associated histone acetyltransferase (HAT) activity, which are separable functions. Acetylation facilitates binding of transcription factors to specific target DNA sequences by destabilizing nucleosomes bound to the promoter region of a target gene (14, 15, 16, 17). The mammalian P/CAF (p300/CBP-associated factor), which contributes the p300/CBP-associated HAT activity, is homologous to the yeast HAT and transcriptional adaptor GCN5 (18). P/CAF inhibited cell cycle progression (18) and promoted myocyte differentiation (19), suggesting P/CAF may also regulate components of the cell cycle.
The eukaryotic cell cycle contains critical genetically conserved components that subserve distinct functions (20, 21). The G1 cyclins are of two classes: the D type cyclins (D1, D2, and D3) and cyclin E. The D type cyclins share structurally conserved motifs that interact with cyclin-dependent kinases 4 and 6 (cdk4/cdk6) and pRB (retinoblastoma protein). The cyclin D1 gene has been implicated in a subset of human tumors including breast and prostate cancer, and overexpression of cyclin D1 has been linked to tumor therapy resistance. Cyclin D1 collaborates with other oncogenes in cellular transformation (22), and cyclin D1 abundance is induced by oncogenic protein including mutants of Ras (23), Rac1 (24, 25), pp60src (26), STAT3 (signal transducer and activator of transcription 3) (27), and ß-catenin/APC mutants (28). The proliferative and oncogenic properties of cyclin D1 may relate to its function as the regulatory subunit of the holoenzyme that phosphorylates and inactivates the tumor suppressor pRB (20, 21). However, cyclin D1 conveys holoenzyme-independent function, inhibiting myocyte differentiation (29) through partially pRB-independent means (30) and binding the estrogen receptor (ER) to induce activity in a ligand-independent manner (31, 32, 33).
In the current studies, we show that cyclin D1 selectively inhibits
ligand-dependent AR function in prostate cancer cells through a
pRB-independent mechanism. The ligand dependency of this interaction
contrasts fundamentally with the previously described
ligand-independent interaction of cyclin D1 with the ER
. Cyclin D1
inhibition of ligand-dependent AR function required a carboxyl terminal
acidic-rich region that is structurally divergent from cyclin D2 and
D3. Cyclin D1 binding to the AR also required the cyclin D1 acidic-rich
carboxyl terminus. The correlation between cyclin D1 binding to the AR
in vitro and the repression of hormone-dependent AR activity
in vivo suggests either a physical interaction between
cyclin D1 and the AR may form a repressor complex or that the AR and
cyclin D1 may compete for a common coactivator. p300 antagonized the
cyclin D1 repressor function, which occurs independently of p300’s
intrinsic HAT activity, and required the associated HAT activity of
P/CAF. Cyclin D1 associated directly with P/CAF. Cyclin D1 and
the AR bound to the same domains of P/CAF. Cyclin D1 repression of
liganded AR activity may involve competition with the AR for binding to
limiting cellular P/CAF.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Dihydrotestosterone
(DHT) induced reporter activity 5-fold, and coexpression of cyclin D1
inhibited this activity by 60% (Fig. 1A). Cyclin D1-mediated
repression of DHT-induced MMTV-LUC reporter activity was observed in
DU145 cells at several different concentrations of DHT (0.1
nM to 100 nM) (Fig. 1B). To examine further the
promoter specificity of cyclin D1-mediated repression, several
different control reporter constructions were examined in DU145 cells
in the presence of the AR and DHT. The activity of the viral promoters
[cytomegalovirus (CMV)-LUC, rous sarcoma virus (RSV)-LUC] and the
luciferase reporter vectors were not regulated by cyclin D1 (Fig. 1C).
Neither cyclin D2 nor cyclin D3 inhibited AR activity (Fig. 1D).
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). Additional reporter genes were examined to determine the
specificity of cyclin D1-mediated repression of DHT- induced AR
activity. Reporter constructions were used encoding immediate early
gene promoters (c-jun LUC, junB LUC), synthetic
response elements (SRE LUC, E2F LUC), and the promoters of cell cycle
control genes (cyclin D1 LUC, cyclin E LUC,
p21Cip1/WAF1 LUC). In contrast with the MMTV-LUC
reporter, cyclin D1 failed to repress the additional promoter reporter
constructions (Fig. 1F). Repression of AR activity was also observed in
293T cell lines (data not shown). These studies suggest that cyclin D1
repression of DHT-induced AR transcriptional activity is promoter
specific.
A Structurally Divergent Acid-Rich Motif in the Carboxyl Terminus
of Cyclin D1 Is Required for Repression of AR Activity
To determine the domains of cyclin D1 involved in repression
of AR activity, expression plasmids encoding mutant cyclin D1 were
constructed and characterized in cultured mammalian cells. The carboxyl
terminus of cyclin D1 diverges structurally from cyclin D2
and D3 (Fig. 2A) due
to the presence of an acid-rich region (residues 272–280). The
expression of the cyclin D1 mutants was assessed in cultured cells by
Western blotting. Sequential deletion of the extreme carboxyl terminus
did not reduce cyclin D1 protein abundance expressed in cultured cells
(Fig. 2B). The abundance of the cyclin D1 mutants was also similar
to wild type cyclin D1 in the presence of DHT (Fig. 2B, lower
panel). Abundance of the carboxyl-terminal deletion mutant (CD1
C241) was increased 2-fold in abundance compared with wild type. To
identify the domains of cyclin D1 required for repression of AR
activity, cotransfection experiments were conducted with either the
wild-type or the mutant cyclin D1 expression vectors. Deletion of the
C-terminal 21 amino acids (CD1 C274) reduced AR repression by 50%
(Fig. 2C). Deletion of an additional three additional residues (CD1
C271) or point mutation of the cyclin D1 carboxyl-terminal LLXXXL
motif (leucines 254/255) abolished AR repression (Fig. 2C). The
repression of DHT-induced AR activity was increased 25% by the
pRB-binding defective mutant (GH) (Fig. 2C). The cdk-binding defective
cyclin D1 mutant (KE) was partially defective in AR repression (Fig. 2C). Therefore, the carboxyl terminus of cyclin D1 is required for full
repression of AR activity. The finding that the pRB-binding domain of
cyclin D1 is not required for repression is consistent with the
observation that cyclin D1 inhibits DHT-induced AR activity in DU145
cells that are pRB deficient.
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) or
glutathione-S-transferase (GST) pull-down assay. 293T cells
were used as they have high transfection efficiency and are AR
deficient. Cells were transfected with expression vectors encoding
either wild-type or mutant AR and cyclin D1 (Fig. 3A) and treated with
DHT or vehicle for 24 h, after which IP was performed on equal
amounts of protein extracts using a cyclin D1-specific antibody
(DCS-11). Western blotting was then performed for the AR and cyclin D1
using specific antibodies. Wild-type AR immunoreactivity was observed
in the cyclin D1 IP (Fig. 3A, lanes 1 and 2) but not in the IP from
cells transfected with the empty expression vector cassette (Fig. 3A, lanes 9 and 10) or from IPs performed with control IgG (data not
shown). AR immunoreactivity was observed in the cyclin D1 IP from cells
transfected with the AR mutants with deleted amino acids 80–93,
96–483, and 707–902 (Fig. 3A, lanes 3–8). The C-terminal mutant AR
1–707 bound cyclin D1 with no alteration in the amount of binding in
response to DHT treatment, consistent with the known deletion of most
of the ligand-binding domain in AR 1–707. These results suggest that
the AR between residues 483 and 707 can bind cyclin D1.
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). The abundance of cyclin D1 in
the pull down was determined by densitometry of the
[35S]-labeled cyclin D1 product. The binding
affinity for each AR fragment was expressed relative to the nonspecific
binding of GST. The GST-AR fragment AR/505–676 bound with high
affinity to cyclin D1 (Fig. 3B). The GST-AR/633–668 bound cyclin D1,
but neither the GST-AR/505–559 nor GST-AR/552–635 bound to cyclin D1,
indicating that AR residues 633–668 are sufficient for cyclin D1
binding.
Experiments were conducted to determine whether cyclin D2 or cyclin D3
was capable of binding to the AR hinge region. GST pull-down analyses
were performed using equal amounts of GST-AR fusion protein and equal
amounts of in vitro translated D type cyclin (Fig. 3C). In
contrast with cyclin D1, neither cyclin D2 nor cyclin D3 bound to
GST-AR when compared with the nonspecific binding of GST.
The Cyclin D1 Acid-Rich Carboxyl Terminus Binds the AR
To identify the region of cyclin D1 required for binding to the
AR, GST pull-down experiments were performed in which in
vitro translated cyclin D1 mutants were incubated with the GST-AR
fragment AR/505–676. Comparison was made to the binding of wild- type
cyclin D1 set as 100%. The mean binding data of three separate
experiments are shown in Fig. 4. Mutation
of the cdk-binding domain did not reduce AR binding. Mutation of
leucines 254 and 255 in cyclin D1 diminished AR binding by only 20%,
and deletion of 21 amino acids from the cyclin D1 C terminus decreased
binding by 64%. The cyclin D1 mutant C267 and other deletions of
the C terminus gave negligible binding to the AR.
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). In
previous studies, the abundance of the mutant p300 proteins was shown
to be similar to the wild type when transfected into mammalian cells.
Overexpression of p300 decreased cyclin D1 repression of MMTV-LUC
activity from 75% repression to 25% (Fig. 5A). To examine candidate
functional domains of p300 required for antagonism of cyclin
D1-mediated AR repression, mutants of the p300 bromo-domain
(1032–1138), the HAT domain (1419–1721), and the CH3 region
(1737–1809) were assessed (Fig. 5A). The p300 CH3 region
1737–1809 mutant failed to rescue cyclin D1-mediated AR
transcriptional repression (Fig. 5A). Deletion of the p300 HAT domain
induced activity further than wild-type p300, suggesting the p300 HAT
domain was not required for rescue.
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)
(34). The abundance of wild-type and mutant P/CAF expressed in cultured
cells was assessed by Western blotting. DU145 cells were transfected
with the Flag-tagged P/CAF expression plasmids, and Western blotting
was performed with the anti-Flag antibody. The P/CAF mutant protein
P/CAF 609–624 was expressed equally to the wild- type protein (Fig. 5B). Cyclin D1-mediated AR repression was antagonized by P/CAF
overexpression (Fig. 5B). The P/CAF HAT domain deletion mutant (P/CAF
609–624), however, failed to rescue and further inhibited
DHT-induced AR activity (Fig. 5B). These studies indicate that P/CAF
HAT activity is required for the reversal of cyclin D1-mediated AR
repression.
Cyclin D1 and P/CAF Binding in Cultured Cells and in
Vitro
We investigated whether cyclin D1 could directly interact with
P/CAF in cultured cells. To determine whether cyclin D1 formed a
complex with P/CAF, 293T cells were transfected with cyclin D1, P/CAF,
and either with or without the AR. IP-Western blotting was then
performed to assess complexes binding to cyclin D1 (Fig. 6). The cyclin D1 IP was immunoreactive
for cyclin D1, AR, and P/CAF (Fig. 6A lanes 1 and 5). The supernatant
(S) was free of cyclin D1 and contained approximately 50% of the
remaining P/CAF (Fig. 6A, lane 2). IP with a control IgG contained
neither cyclin D1 nor P/CAF (Fig. 6A, lane 3). AR Western blotting
demonstrated AR in the cyclin D1 IP and in the supernatant (Fig. 6A, lanes 1 vs. 2). Approximately 35% of the total AR was bound
to cyclin D1 and approximately 50% of the cellular P/CAF was bound to
cyclin D1.
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lanes 5 vs. 6). This finding suggests the decrease in P/CAF
binding to cyclin D1 upon the addition of DHT is likely due to the
presence of the AR.
To determine whether the AR binds to cyclin D1 or P/CAF in
vivo, IP-Western blotting was performed using extracts from murine
liver with comparison made to direct Western blotting of the
hepatocellular extracts (Fig. 6C, lane 2 vs. 1). The AR
antibody immunoprecipitated the AR (Fig. 6C, lane 2) and no
immunoreactive AR band was observed in control IgG (not shown). The AR
IP was also immunoreactive for cyclin D1 and for P/CAF. These studies
demonstrate that the cyclin D1-AR interaction observed in cultured
cells is also observed in tissues.
As these studies showed P/CAF can be immunoprecipitated in association
with cyclin D1, it remained to be determined whether cyclin D1 could
interact directly with specific domains of P/CAF. Several
transcriptional activators, viral proteins, and coactivators have been
shown to interact directly with P/CAF. GST pull-down experiments were
therefore conducted with affinity-purified P/CAF and GST cyclin D1 or
GST alone as a control (Fig. 7). The
amount of P/CAF protein was confirmed by Western blotting using the
Flag epitope (Fig. 7, upper panel). Incubation of
affinity-purified P/CAF with GST alone showed no interaction (Fig. 7, middle panel). Incubation of affinity-purified P/CAF with
GST-cyclin D1 showed strong binding (>20% of the input). The P/CAF
internal deletion mutants of the HAT (511–656), Ada2 homology
region (655–701), and bromo domain (729–832) were defective in
binding to cyclin D1. In addition, the internal deletion mutant
5–122 was defective in binding to cyclin D1. The domains of P/CAF
required for binding to cyclin D1 resemble the domains required for
binding to the AR (35), raising the possibility that cyclin D1 may
inhibit AR function by binding to the same sites on P/CAF.
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). Western blotting of the P/CAF IP
using a GST antibody demonstrated the presence of the
GST-AR505-676 fusion
protein (Fig. 8, lower panel) indicating P/CAF and AR are
capable of interacting in a pull-down assay. The preaddition of excess
GST-cyclin D1 to the mix of Flag-tagged P/CAF and
GST-AR505-676 fusion
protein abolished the presence of immunodetectable
GST-AR505-676 in the P/CAF
IP (Fig. 8, lane 4, lower panel). The preaddition of equal
molar amounts of GST protein did not interfere with the binding of
P/CAF to GST-AR (Fig. 8 lane 2, lower panel). GST-cyclin D1
was observed in the supernatant by Western blotting in conjunction with
AR505-676 (Fig. 8, lane
3). These studies suggest that cyclin D1 has the
capacity to inhibit the association between P/CAF and the AR in
vitro.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(ER). In
contrast with the current studies in which cyclin D1 selectively
inhibited liganded AR activity, it is the activity of the unliganded
ER that is induced by cyclin D1 (31, 36). Although cyclin D1 binds
to ER in cultured cells, it is not known which residues of either
ER or of cyclin D1 are required for direct interaction (37). The
carboxyl-terminal LXXXLL motif of cyclin D1 plays a role in cyclin
D1-mediated induction of basal ER activity through recruiting SRC-1
(36). Although the ER is capable of binding to cyclin D1 (31, 36)
and P/CAF (38), the domains of P/CAF binding to the ER are distinct
from the regions binding to the AR (data not shown). Thus, the domains
of P/CAF bound by the nuclear receptor may determine functional
interactions with cyclin D1.
The association between cyclin D1 and the AR demonstrated by IP-Western
blotting of cultured cells and using in vitro analysis
suggests the basic region of the AR interacts with the carboxy-terminal
region of cyclin D1. The domains of cyclin D1 required for binding to
the AR correlated well with the regions required for transcriptional
repression of the ligand-bound AR. Cyclin D2 and D3 neither repressed
ligand-induced AR activity nor bound to this region of the AR in GST
pull-down experiments. This finding suggests that the structural
divergence among the D type cyclins in this region may abrogate
critical determinants for AR binding. The C274 mutant was 50%
defective in repression of DHT-induced MMTV-LUC activity and bound AR
with 35% of wild-type binding affinity on GST pull-down analyses (Fig. 2C). The region of cyclin D1 from 267 to 274 was absolutely required
for both binding to the AR and for repression of liganded AR activity.
Alignment of cyclin D1 and structural comparison with cyclin A suggests
the acid-rich region (267 PKAAEEEE 271) and leucine-containing motif
(LLXXXL), required for AR binding, align at the C terminus of helix 5.
The structure of cyclins A and H deduced through crystallization
reveals a core of two repeats containing five -helical bundles that
form the cyclin fold (39). The likely amphipathic -helical
structure of cyclin D1 within the AR interaction domain may serve
as an interaction domain with other proteins. The region of the AR
involved in direct binding to cyclin D1 (residues 633–668) contains
predominantly basic residues, suggesting an ionic basis for binding to
cyclin D1.
In the current studies deletion of the p300 CH3 domain abolished relief
of cyclin D1-mediated AR transrepression. Both p300 and P/CAF have
intrinsic HAT function. Deletion of the P/CAF HAT domain, but not the
p300 HAT domain, abolished its ability to relieve cyclin D1-mediated AR
repression. As the p300 CH3 region is capable of binding P/CAF, it is
likely that the p300 CH3 mutant is defective through failed recruitment
of P/CAF. The finding that P/CAF HAT activity, but not p300 HAT
activity, was required for relief of cyclin D1-mediated AR
transrepression is consistent with recent observations in which the
acetyltransferase activities and substrate specificities of p300 and
P/CAF were shown to be distinguishable (40). These findings are also
consistent with previous observations that the functional activities of
p300 and P/CAF are restricted to specific activators or promoters (17, 41). In transfection assays, ligand-induced AR activity was inhibited
by cyclin D1 and enhanced by P/CAF. The mutual antagonism between
cyclin D1 and the AR may be due to competition for binding to a common
intermediary protein or a limiting cofactor such as P/CAF. In support
of a model in which cyclin D1 and AR may compete for a common
intermediary protein are the in vitro findings that cyclin
D1 and the AR bound to similar regions of P/CAF (Fig. 7) and that the
preaddition of GST cyclin D1 fusion protein inhibited the binding of
P/CAF to the AR (Fig. 8). Support for the biological relevance of the
association between cyclin D1 and the AR include our findings that the
AR was associated with cyclin D1 and P/CAF in vivo, and in
transfected cells using IP-Western blot analysis (Fig. 6). Furthermore,
the associations between cyclin D1 and the AR appear to be hormone
regulated. Thus, in cultured cells, the amount of P/CAF associated with
cyclin D1 was reduced by the presence of liganded AR. Together, these
studies suggest that cyclin D1 inhibition of liganded AR activity may
be due to mutually exclusive binding of cyclin D1 and AR for P/CAF. As
P/CAF forms a multiprotein complex, it is likely that additional
components regulating this interaction in vivo remain to be
determined. As the domain of P/CAF that binds to cyclin D1 overlaps the
Twist binding domain (42), it will be of interest to
determine whether the differentiation function of Twist or
other basic helix-loop-helix proteins are also regulated by cyclin
D1.
The current studies, however, provide evidence that cyclin D1 can selectively and specifically inhibit liganded AR activity. Cyclin D1 can inhibit differentiation and either promote or inhibit cellular proliferation in a cell type-specific manner (reviewed in Ref. (43). The AR also has both differentiation and proliferation functions. For example, the DHT-liganded AR plays an important role in the induction of male secondary sexual characteristics (3). Thus, AR gene mutations described in complete androgen insensitivity syndrome causes XY genotypic males to develop as phenotypic females due to defective AR function (44). In cultured prostatic cells, DHT can induce differentiation, as evidenced by the cell cycle arrest and increased expression of enzymatic differentiation markers (45) or proliferation in cell lines with mutant AR (46). Because of the functional redundancy between components of the cell cycle-regulatory apparatus, it may be difficult to dissect the independent contribution of cyclin D1 to liganded AR activity in vivo. The AR is located in a broad array of tissues and contributes to diverse signal transduction pathways in breast, brain, and prostate, and the role of cyclin D1 in regulating signal transduction in these tissues remains to be determined.
Cyclin D1 binding to P/CAF in vitro required the HAT domain of P/CAF. Mutational analysis of the Gcn5p HAT domain identified four subdomains with catalytic activity mediated through the carboxyl-terminal subdomains (47). Mutation within subdomain IV of Gcn5p abolished HAT activity in vitro (47). In the current studies an expression plasmid encoding a deletion within the HAT domain abolished rescue of cyclin D1 repression of liganded AR activity. The carboxyl-terminal bromodomain of P/CAF, which forms a four-helix bundle that interacts specifically with acetylated lysine (48), remains intact in this mutant and may therefore reduce AR activity by competing with endogenous P/CAF. Although speculative at this time, our studies predict that the binding of cyclin D1 to the HAT domain of P/CAF may regulate other functions of P/CAF. P/CAF HAT activity contributes to regulation of transcription, inhibition of cell cycle progression, and the induction of differentiation (18, 41). The cyclin D1 gene product, in contrast, can inhibit differentiation and promote cell cycle progression in several cell types. P/CAF is associated with at least 20 polypeptides in cultured cells including the TBP-associated factors (TAFs), which are subunits of transcription factor IID (TFIID), and other TAFs (49). The association of P/CAF with these polypeptides and the direct HAT activity conveyed by P/CAF may both contribute to the regulation of AR function. P/CAF has the capacity to augment gene expression both by acetylating core histones and also by directly acetylating a subset of transcription factors including p53 (50) and the AR (35) reviewed previously (41). The contribution of cyclin D1 to other functions of P/CAF remains to be explored.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1737–1809, 1419–1721
(15), pCMVHAbromop300, which deletes residues 1,032–1,138 of p300,
rP/CAF, P/CAF 609–624 (52), were previously described. The
reporter plasmids c-fos LUC, c-Jun LUC, JunB LUC,
SRELUC, E2F LUC, cyclin D1 LUC, cyclin E LUC,
p21Cip1/WAF1 LUC, CMV LUC, and RSV LUC were
previously described (23, 53, 54, 55). The human cyclin D1 mutants were
derived by PCR-directed amplification using sequence-specific primers
and cloned into pRc/CMV, and the integrity of all constructs was
confirmed by sequence analysis. The AR expression vectors, ARWt,
AR80–93, AR96–483, and AR 1–707 (56), and fusion proteins,
GST-AR/676–844, GST-AR/676–919, GST-AR/505–919, GST-AR/505–676,
GST-AR/505–559, GST-AR/552–635, and GST-AR/633–668 (57), the
reporter MMTV-LUC (from Dr. R. Evans), and the Flag-tagged P/CAF
mutants (42) were previously described. GST-cyclin D2 and GST-cyclin D3
were gifts from Dr. M. Pagano. Cell culture, DNA transfection, and luciferase assays were performed as previously described (53, 54). A CMV-Renilla luciferase plasmid, pRL-CMV (Promega Corp., Madison, WI), was included to control for transfection efficiency as indicated. Renilla luciferase activity was assayed according to the manufacturer’s instructions for the Dual-Luciferase Reporter Assay System. The prostate cancer cell lines PC3 and DU145 (ATCC, Manassas, VA) and the 293T cell line were cultured in DMEM with 10% charcoal-stripped FCS, 1% penicillin, and 1% streptomycin. Cells were transfected by calcium phosphate precipitation or lipofection using lipofectamine PLUS (Life Technologies, Inc., Gaithersburg, MD), the medium was changed after 6 h or 3 h, respectively, and luciferase activity was determined after 48 h. At least two different plasmid preparations of each construct were used. In cotransfection experiments, a dose-response was determined in each experiment with 300 ng and 600 ng of expression vector and the promoter reporter plasmids (2.4 µg). Luciferase activity was normalized for transfection using the renilla luciferase as internal control as previously described. Luciferase assays were performed at room temperature using an Autolumat LB 953 (EG&G Berthold) as previously described (53). The -fold effect was determined for 300–600 ng of expression vector with comparison made to the effect of the empty expression vector cassette, and statistical analyses were performed using the Mann-Whitney U test.
Western Blots
The antibodies used in Western blot analysis were a monoclonal
cyclin D1 antibody DCS-6 (NeoMarkers Lab Vision Corp., Fremont, CA), a
rabbit anti-P/CAF antibody (18), an -guanine nucleotide dissociation
inhibitor (GDI) (58) (a gift from Dr. Perry Bickel, Washington
University St. Louis, MO, used as internal control for protein
abundance), anti-M2 Flag antibody (Sigma, St. Louis, MO),
and antibodies from Santa Cruz Biotechnology, Inc., (Santa
Cruz, CA): cyclin D1 antibody (HD11), GST (B-14), and AR (N-20). For
detection of cyclin D1, the membrane was incubated with antimouse
cyclin D1 antibody DCS-6 (NeoMarkers Lab Vision Corp.), washed with
0.05% TPBS three times, and then incubated with horseradish
peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and washed again. An
antirabbit horseradish peroxidase-conjugated secondary antibody was
used for AR (1:3,000), and an antimouse horseradish
peroxidase-conjugated secondary antibody was used for the Flag epitope
(1:2,000). Proteins were visualized by the enhanced chemiluminescence
system (Amersham Pharmacia Biotech, Arlington Heights,
IL). The abundance of immunoreactive protein was quantified by
phosphoimaging using a computing densitometer (Image Quant version
1.11, Molecular Dynamics, Inc., Sunnyvale, CA).
Protein Interaction Assays in Vitro and in Cultured
Cells and in Vivo
In vitro
[35S]methionine-labeled proteins were prepared
by coupled transcription-translation with a TNT coupled reticulocyte
lysate kit (Promega Corp., Madison, WI), using 1.0 µg of
plasmid DNA in a total of 50 µl. Flag-tagged P/CAF proteins were
expressed in Sf9 cells by infecting with recombinant baculovirus and
were purified as described (15). GST fusion proteins were prepared from
Escherichia coli cells as described previously (42). GST
fusion proteins were induced for 4 h at 30 C with 0.1
M
isopropyl-ß-D-thiogalactopyranoside, and crude
lysates were prepared at 4 C. Cell pellets were spun down and
resuspended in 15 ml PBS containing 2 mM
dithiothreitol (DTT), 1 mM
phenylmethylsulfonylfluoride (PMSF), 1 µg/ml pepstatin, 1 µg/ml
leupeptin, and 1 µg/ml aprotinin. Lysozyme (2 mg/sample) was added
and incubation performed on ice for 10 min. Samples were sonicated
using 20-sec pulses until lysed. One milliliter of 15% Triton X-100 in
PBS was added, and the samples were then centrifuged at 40,000 rpm for
15 min at 4 C. The supernatant was added to 500 µl of glutathione
sepharose beads (Amersham Pharmacia Biotech), and the
mixture was rotated at 4 C overnight. The supernatant was discarded and
the pellet washed three times in PBS containing 100
mM DTT and 1% Tween 20, and three times in PBS
containing 50 mM Tris-HCl and 5
mM DTT (pH 8.0). The pellet was resuspended in
400 µl of this buffer and 50 µl of glutathione (100
mM in Tris HCl, pH 8.0) were added. Elution of
proteins was performed for 1 h, and beads were separated by
centrifugation. Glutathione was removed by dialysis at 4 C in 20
mM Tris, 25 mM NaCl, and 2
mM EDTA.
In vitro protein-protein interactions were performed as described (31). The in vitro translated protein (15 µl of cyclin D1) was added to 5 µg of GST or equal amounts of GST-AR, in 225 µl of binding buffer (50 mM Tris-HCl, 120 mM NaCl, 1 mM DTT, 0.5% NP-40, 1 mM EDTA, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 2 µg/ml pepstatin) and rotated for 2 h at 4 C. Fifty microliters of glutathione-sepharose bead slurry were added, and the mixture was rotated for a further 30 min at 4 C. Beads were washed five times with 1 ml of binding buffer and 30 µl of binding buffer were added after the final wash. Baculovirus- expressed Flag-tagged P/CAF (1 µg) was used in pull-down experiments as previously described (35). In competition experiments baculovirus P/CAF (1 µg) was incubated with GST-AR505-676 (1 µg), and IP was performed with the M2 (Flag) antibody. Competition was performed through preincubation with GST-cyclin D1 (1 µg) or GST (1 µg) alone.
IP Western blot analysis was performed with murine hepatic tissue (as previously described) (59) or 293T cells. 293T cells transfected with pSV-AR, pCMV-cyclin D1, pCI-P/CAF, or expression vector control were treated for 36 h with 10-7 M DHT or vehicle as control. Cells were rinsed with PBS, harvested by scraping, pelleted, and lysed in buffer (50 mM HEPES, pH 7.2, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1% Tween 20, 0.1 mM PMSF, 2.5 µg/ml leupeptin, 0.1 mM sodium orthovanadate (Sigma). Extracts were cleared by centrifugation and further precleared by rocking at 4 C with washed protein A Sepharose (Roche Molecular Biochemicals, Indianapolis, IN). Five hundred micrograms of precleared extract were immunoprecipitated with 1 µg of cyclin D1 antibody (DCS-11, NeoMarkers Lab Vision Corp.) or AR antibody (N-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or equivalent amounts of appropriate control IgG (Santa Cruz Biotechnology, Inc.) and 50 µl of protein A sepharose for 8–12 h at 4 C. Beads were washed five times with lysis buffer and boiled in SDS sample buffer, and released proteins were resolved by 10% SDS-PAGE. The gel was transferred to nitrocellulose and Western blotting was performed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by an award from the Pfeiffer Foundation, RO1CA70897 and RO1CA75503 (to R.G.P.), NIH Grants CA-70297 (Z.J.S.), and American Cancer Society Grant RPG98213 (Z.J.S.). Work conducted at the Albert Einstein College of Medicine was supported by Cancer Center Core NIH Grant 5-P30-CA13330–26.
1 These authors contributed equally to the manuscript. 
Received for publication September 11, 2000. Revision received January 9, 2001. Accepted for publication January 30, 2001.
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