MOLECULAR AND CELLULAR BIOLOGY, Dec. 2003, p. 8563–8575
0270-7306/03/$08.00?0 DOI: 10.1128/MCB.23.23.8563–8575.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 23, No. 23
Acetylation of Androgen Receptor Enhances Coactivator Binding and
Promotes Prostate Cancer Cell Growth
Maofu Fu,1Mahadev Rao,1Chenguang Wang,1Toshiyuki Sakamaki,1Jian Wang,1Dolores Di Vizio,1
Xueping Zhang,1Chris Albanese,1Steven Balk,2Chawnshang Chang,3Saijun Fan,1Eliot Rosen,1
Jorma J. Palvimo,4Olli A. Ja ¨nne,4Selen Muratoglu,5Maria Laura Avantaggiati,5and
Richard G. Pestell1*
Department of Oncology, Lombardi Cancer Center, Georgetown University, Washington, D.C. 200571; Hematology-Oncology
Division, Beth Israel Deaconess Medical Center, Boston, Massachusetts 022152; George Whipple Laboratory for Cancer
Research, Departments of Urology, Pathology, Radiation Oncology, Biochemistry, and Toxicology, and Cancer Center,
University of Rochester, Rochester, New York 146423; Biomedicum Helsinki, Institute of Biomedicine, and
Department of Clinical Chemistry, University of Helsinki, FIN-00014 Helsinki, Finland4; and
Department of Pharmacology, George Washington University Medical Center,
Washington, D.C. 200375
Received 7 April 2003/Returned for modification 20 May 2003/Accepted 27 August 2003
Modification by acetylation occurs at ?-amino lysine residues of histones and transcription factors. Unlike
phosphorylation, a direct link between transcription factor acetylation and cellular growth or apoptosis has not
been established. We show that the nuclear androgen receptor (AR), a DNA-binding transcriptional regulator,
is acetylated in vivo. The acetylation of the AR is induced by ligand dihydrotestosterone and by histone
deacetylase (HDAC) inhibitors in living cells. Direct AR acetylation augmented p300 binding in vitro. Con-
structs mimicking neutral polar substitution acetylation (ARK630Q, ARK630T) enhanced p300 binding and
reduced N-CoR/HDAC/Smad3 corepressor binding, whereas charged residue substitution (ARK630R) reduced
p300 binding and enhanced corepressor binding. The AR acetylation mimics promoted cell survival and growth
of prostate cancer cells in soft agar and in nude mice and augmented transcription of a subset of growth control
target gene promoters. Thus, transcription factor acetylation regulates coactivator/corepressor complex bind-
ing, altering expression of specific growth control genes to promote aberrant cellular growth in vivo.
Prostate cancer is the second leading cause of cancer death
in American males. Although potentially curable by radical
prostatectomy or radiation therapy, metastatic disease is com-
mon at presentation and may occur subsequently in patients
treated with curative intent. The androgen receptor (AR) is a
classical nuclear receptor (NR) that binds testosterone and is
required for the induction of male secondary sexual character-
istics. The AR conveys several dissociable functions, including
transactivation, transrepression, growth regulation, basal activ-
ity, and context-dependent cell survival or apoptosis functions.
Aberrant AR function plays an important role in prostate
cancer (1). The wild-type AR can induce cellular differentia-
tion or cellular apoptosis in prostate cancer cells (2, 16, 47).
Both AR-dependent and AR-independent mechanisms con-
tribute to prostate cancer cellular growth. Somatic missense
AR gene mutations have been detected in prostate cancer cell
lines, xenografts, and primary and metastatic forms of prostate
The NR superfamily, of which the AR is a member, are
transcriptional regulators that coordinate important metabolic
and differentiation functions (29, 42). Transcriptional activity
of NR is regulated by ligand-dependent recruitment of coac-
tivator/corepressor proteins. In the presence of ligand, the
most carboxyl-terminal helix 12 (H12) of NR folds over the
ligand-binding hydrophobic pocket, creating structural sur-
faces that bind the basal transcriptional apparatus and recruit
coactivators required for efficient transactivation (29, 42).
Corepressor function involves interaction with SIN3 and a
histone deacetylase (HDAC) function (3, 15, 30). Several find-
ings suggest a role for corepressor proteins with HDAC activity
in at least one component of the AR’s functions. Liganded AR
transcriptional activity is induced by trichostatin A (TSA) (7),
the HDAC binding protein Smad3 inhibited liganded AR ac-
tivity (7, 8, 13), HDAC1 bound to the AR in vivo, and HDAC1
binding to the AR was dissociated by the ligand dihydrotest-
osterone (DHT) (7). In the presence of hormone, corepressors
dissociate from the NR. As corepressor and coactivators show
substantial overlap in their site of binding to the NR (19),
coactivator recruitment may be contingent upon corepressor
disengagement (29, 42).
The cointegrator proteins p300 and CBP augment NR ac-
tivity through several functions. They recruit coactivators and
serve as a molecular bridge between the NR and the basal
transcription apparatus. The cointegrator proteins p300/CBP
augment NR activity, in part related to their intrinsic histone
acetyltransferase (HAT) activity (31, 37, 38). The intrinsic
HAT activity acetylates both histones and nonhistone proteins
to regulate their activity (37). Histone acetylation may contrib-
ute to nucleosome destabilization, facilitating transcription
factor binding to specific target DNA sequences in the pro-
moter region of a target gene (31, 38). Direct acetylation of
* Corresponding author. Mailing address: Department of Oncology,
Lombardi Comprehensive Cancer Center, Research Building, Room
E501, 3970 Reservoir Rd., NW, Box 571468, Georgetown University,
Washington, DC 20057. Phone: (202) 687-2100. Fax: (202) 687-4638.
lysine residues in either histone or transcription factors neu-
tralizes the residue’s positive charge and increases its polarity,
events that are mimicked by a glutamine substitution. The AR
is directly acetylated, and mutation of the residues acetylated
in vitro abrogates acetylation of the full-length AR in living
cells (7). Although loss-of-function mutation of transcription
factor lysine residues implicate acetylation in transcription fac-
tor activity, the physiologic effect of transcription factor acet-
ylation in regulating cellular growth remains to be determined
(4, 45). The current studies show for the first time that AR
transcription factor acetylation regulates resistance to thera-
peutic agents and determines prostate cell growth in vivo.
MATERIALS AND METHODS
Reporter genes and expression vectors. The androgen-responsive synthetic
reporter constructions MMTV-Luc and PSA-Luc, cyclin D1-Luc, cyclin E-Luc,
p21Cip1-Luc, cdc25A-Luc and the expression vectors pCMVHA-p300, HA-
TIP60, TIP60?HAT, pSG5SRC-1a, pSG5ARA70, pSG5ARA55, and hARwt
(pcDNA3) were described previously (7, 8, 36). ARK630Qand ARK630Twere
derived by PCR-directed amplification and cloned into pcDNA3. The expression
plasmids encoding pFlag-CMV-2-Ubc-9, Flag-Smad3, Smad3?C, pCEP4-
MEKK1wt, pCMX-FlagN-CoR, pCMV-HDAC1, and pCMV-EGFP were de-
scribed previously (7, 8).
Cell culture, DNA transfection, chemicals, and luciferase assays. The reporter
assays, cell culture, DNA transfection, and luciferase assays were performed as
previously described (7). The prostate cancer cell line DU145 and the 293 T-cell
line were cultured in Dulbecco’s modified Eagle’s medium supplemented with
10% fetal bovine serum, 1% penicillin, and 1% streptomycin. Cells were incu-
bated in medium containing 10% charcoal-stripped fetal bovine serum prior to
experimentation with dihydrotestosterone (DHT) (7). Statistical analyses was
performed with the Mann-Whitney U test.
Apoptosis, colony formation, soft agar assays, and nude mouse tumor implan-
tation. Apoptosis was detected by morphological analysis of green fluorescent pro-
tein (GFP)-transfected cells (7, 8). At least 200 cells were counted with a fluorescent
microscope, and cells were scored for blebbing and chromatin condensation by an
investigator blinded to the experimental condition. Stable cell lines were assessed for
cellular proliferation and colony formation. We seeded 2 ? 105DU145 cells into
six-well plates and transfected them 24 h later with 2.5 ?g of wild-type AR, AR
acetylation mutants, or the control vector, pcDNA3. Forty-eight hours after trans-
fection, the cells were seeded into a 15-cm plate, and G418 (2 mg/ml) was included
in the medium for selection of the transfected cells. Single colonies were harvested
on day 14 and maintained in Dulbecco’s modified Eagle’s medium supplemented
with 10% fetal bovine serum and 500 ?g of G418 per ml.
AR expression was determined by Western blotting, and three independent
colonies for wild-type AR and each of the AR mutants were selected. Anchor-
age-independent growth of stable cell lines was assayed by soft agar growth and
in nude mice. Apoptosis was determined within tumors of similar volumes from
nude mice assessed by terminal deoxynucleotidyltransferase-mediated dUTP-
biotin nick end labeling (TUNEL) staining (ApopTag Red in situ apoptosis
detection kit; Intergene, Purchase, N.Y.), evaluating five fields at 40?, counting
at least 400 cells per field. The tetrazolium salt (MTT) assay was performed as
previously described (8).
Western blots, immunoprecipitation, and immunoprecipitation-HAT assays.
Antibodies used for Western blot analysis and immunoprecipitation as previ-
ously described include anti-AR (N-20), anti-p300 (Santa Cruz), anti-M2-Flag
(Sigma), anti-acetyl-lysine (Upstate Biotech), anti-acetyl-lysine motif p53320(12,
17, 27, 28), anti-HDAC1 (Upstate Biotech), and guanine disassociation inhibitor
(GDI) as a protein-loading control (36). Immunoprecipitation-Western blot
analysis was performed with DU145 stable cell lines expressing the wild-type AR
FIG. 2. AR acetylation in vivo. (A) The cell lysate was extracted
from wild-type male liver and subjected to immunoprecipitation (IP)
with either normal rabbit IgG, anti-AR, or anti-acetyl-lysine antibody.
The immunoprecipitate was separated by SDS-PAGE and blotted with
anti-AR antibody (Upstate Biotech). (B) The mouse liver lysate was
also immunoprecipitated with normal rabbit IgG or anti-AR antibody
and blotted with anti-acetyl-lysine antibody. (C, E) The DU145ARwt
stable cell lines were treated with vehicle (ethanol) or DHT (100 nM)
for 12 h and then treated with TSA (30 nM) for 6 h. Immunoprecipi-
tation was done with anti-AR or anti-acetyl-lysine peptide (p53320) as
indicated. Prior to immunoprecipitation with anti-acetyl-p53320, cell
lysates were first cleared with an anti-p53 antibody (anti-p53[1-393];
sc-4246; Santa Cruz Biotech). The immunoprecipitate was resolved by
SDS-PAGE and blotted with anti-AR antibody. (D, F) Western blot
(WB) of cell lysates precleared with anti-p53 antibody (sc-4246).
FIG. 1. AR acetylation site regulates ligand sensitivity and specificity. (A) The AR acetylation motif indicating lysine 630 (?), with homology
shown to the acetylation motif of human p53 and ACTR proteins. (B) In vitro histone acetyltransferase (HAT) assays used wild-type AR peptides
incubated with baculovirus-purified p300 and [14C]acetyl coenzyme A. (C) Activity of the PSA-Luc and (D) MMTV-Luc reporters was assessed
in DU145 cells with the wild-type and mutant ARs and is shown as relative luciferase activity (mean ? standard error of the mean for n ? 6
separate transfections). (E) The expression plasmids encoding wild-type AR or AR mutants were transfected and examined for DHT dose
responsiveness in DU145 cells with the MMTV-Luc reporter. Cells were treated with either vehicle or DHT (24 h). (F) The wild-type AR and AR
acetylation site mutants were assessed for responsiveness to flutamide with induction shown compared to equal amounts of the empty expression
vector cassette (pSG5). The data are means ? standard error of the mean for n ? 9. (G) The wild-type AR and AR acetylation site mutants were
transfected into 293T cells, treated with 100 nM DHT or 10 nM flutamide for 24 h. The cell lysates were then subjected to Western blotting with
the antibodies to the AR or GDI as the protein-loading control. (H) The wild-type AR and AR acetylation site mutants were transfected into 293T
cells, treated either with vehicle, DHT (100 nM), or TSA (100 nM) for 24 h. Western blotting of the nuclear and cytoplasmic fractions is shown.
VOL. 23, 2003ANDROGEN RECEPTOR ACETYLATION8565
FIG. 3. Direct AR acetylation enhances p300 binding. (A, B, and D) Extracts from cells transfected with the AR expression vectors were
subjected to Western blot analysis (A) or AR immunoprecipitation Western blotting (B and D) to detect p300 and the AR. The AR immuno-
precipitation supernatant (SN) was immunoblotted for p300 to show the proportion of p300 not bound to the AR. (C) The relative binding of the
AR mutants to p300 were shown as mean ? standard error of the mean (n ? 3). (E) Acetylation of the AR enhances p300 binding in vitro. GST
pull-down was performed with p300 and either acetylated AR (lanes 1 to 3) or unacetylated AR (lanes 4 to 6). Western blotting of the GST-AR
pull-down product is shown. (F) p300 in vitro HAT assay. Baculovirus-expressed p300 protein (100 ng) was incubated with either GST or
GST-AR624-676in the presence of [14C]acetyl coenzyme A, resolved by SDS-PAGE, and exposed to a phosphoimaging screen for 24 h. The
autoradiograms of the14C-acetylated p300 and AR fusion protein are indicated. (G and H) Increased p300-mediated trans-activation of AR
acetylation mutants. The MMTV-Luc reporter was assessed with the wild-type AR and AR acetylation site mutants, with p300 or equal amounts
of the empty expression vector cassettes (pCMV), and treated with vehicle or DHT (10?7M) for 24 h. The data shown were normalized to the
DHT-induced activity for the AR construct (mean ? standard error of the mean, n ? 9 separate transfections).
8566 FU ET AL.MOL. CELL. BIOL.
or 293T cells transfected with pcDNA3AR, pcDNA3ARK630Q, pcDNA3ARK630T,
pCMV-HA-p300, pCMX-FlagN-CoR, or the expression vector control (7, 8, 36).
The DU145ARwt stable cells were treated with vehicle (ethanol) or DHT (10 nM)
for 12 h, TSA (1 ?M) or suberoylanilide hydroxamic acid (SAHA, 5 ?M; Aton
Pharma Inc.) for 6 h. The cell lysates were harvested and subjected to immunopre-
cipitation as shown in the figures.
For immunoprecipitation with anti-acetyl-p53320, the cell lysates were pre-
cleared with anti-p53 antibody (p53 Pab240; Santa Cruz Biotech), and the su-
pernatants were subjected to immunoprecipitation with anti-acetyl-p53320. In
vitro acetylation assays were performed with baculovirus-purified p300 as a
source for HAT and AR peptides as previously described (7). Glutathione
S-transferase (GST) pull-down experiments with the acetylated AR and p300
were conducted as described (33). The AR fusion protein was expressed from
GST-AR624-676as it is efficiently acetylated by p300 (7), incubated with 1 mM
acetyl-coenzyme A, and pull-down was conducted with p300. The Western blot-
ting for the AR associated with p300 was conducted with the GST antibody.
In vivo acetylation. Murine hepatocellular lysate were extracted in cell lysis
buffer (20 mM HEPES [pH 7.5], 0.1 M KCl, 0.4 mM EDTA, 0.2% NP-40, 10 mM
?-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 10 ?g of pepstatin
per ml, 1 ?g of NaVO4per ml, 5 mM sodium butyrate, 50 ?M TSA) and
immunoprecipitated with normal rabbit IgG, anti-AR, or anti-acetyl-lysine anti-
body. The immunoprecipitate was separated by sodium dodecyl sulfate-polyac-
rylamide gel electrophoresis (SDS-PAGE) and blotted with either anti-AR or
anti-acetyl-lysine antibodies (7).
AR acetylation site governs ligand sensitivity and specificity.
The AR is acetylated in cultured cells, requiring an acetylation
motif (KxKK) which is conserved with other NR and with the
tumor suppressor p53 (7, 37) (Fig. 1A). A peptide containing
the AR acetylation motif was sufficient for acetylation by p300
(Fig. 1B). Of the three lysine residues within the motif, we
chose to study ARLys630in more detail. An AR lysine (Lys630-
Ala) substitution was most defective in ligand-induced trans-
activation and abrogated AR acetylation in cultured cells (7).
The acetylated ARK630residue was replaced with glutamine
(Lys630-Glu) to create an acetylation mimic. Since acetylation
of lysine neutralizes its positive charge and increases its hydro-
phobicity, comparison was made to an AR containing a polar
uncharged threonine residue substitution of ARK630, a somatic
mutation that occurs in prostate cancer patients.
Transcriptional activity of the ARK630Qwas greater than
that of wild-type AR with either the androgen-responsive pros-
tate-specific antigen gene (PSA) promoter (wild-type AR 4.5-
fold, ARK630Q10-fold, and ARK630T15-fold) (Fig. 1C) or
androgen-responsive mouse mammary tumor virus (MMTV)-
luciferase (Luc) reporter gene (wild-type AR 4.3-fold,
ARK630Q10-fold, and ARK630T14-fold; P ? 0.01) (Fig. 1D).
The AR acetylation site mimic was more active at lower DHT
concentrations than the wild-type AR (Fig. 1E) (at 0.1 nM
DHT, wild-type AR 1.5-fold, ARK630T2.5-fold; at 1 nM DHT,
wild-type AR 1.8-fold, ARK630Q4.5-fold, and ARK630T5-fold).
The DHT antagonist flutamide antagonized DHT-induced
wild-type AR activity; however, the AR acetylation mimic was
relatively resistant (Fig. 1F).
In order to determine the mechanism of enhanced ligand-
dependent transactivation by ARK630Qand ARK630Tof the
androgen-responsive reporter genes, we examined the relative
abundance of the AR wild-type and acetylation mutants. In
AR-deficient 293T cells, transient transfection of the AR ex-
pression vector and subsequent Western blot analysis demon-
strated that the relative abundance of AR was similar for the
AR wild type and the acetylation mutant in the presence of
DHT or flutamide (Fig. 1G). In addition, the relative distribu-
tion of the wild type and acetylation mimic mutant AR be-
tween the nuclear and cytoplasmic fractions was similar in the
presence of either DHT or TSA (Fig. 1H). Together, these
studies suggest that the AR acetylation mutants are expressed
similarly to wild-type AR in cultured cells and are subcellularly
distributed like the AR wild type.
AR is acetylated in vivo. To determine whether the AR is
acetylated in living cells in vivo, immunoprecipitation of mu-
rine hepatic tissue was performed with antibodies to either the
AR or acetylated lysine residues, with subsequent Western
blotting for the AR. The anti-acetyl-lysine antibody efficiently
precipitated the AR (Fig. 2A). In the reciprocal immunopre-
cipitation with the AR antibody, Western blotting with the
anti-acetyl-lysine antibody demonstrated AR immunoreactiv-
ity (Fig. 2B). In view of the conservation of the AR acetylated
lysine motif with the acetylated p53 lysine motif (7), we used
previously well characterized antibodies generated to an acety-
lated lysine motif peptide of p53 (27) in immunoprecipitation
studies of stable DU145-AR cell lines. Subsequent to clearing
of p53, the anti-acetyl-lysine-p53 antibody immunoprecipitate
was subjected to Western blot for coprecipitated AR (Fig. 2C).
The amount of AR precipitated by antibody to the acetylated
lysine motif was increased by the addition of either DHT or
TSA (Fig. 2C, lane 2 versus 4 and 6).
Western blotting for AR expression in the DU145-AR
wild-type stable cell line demonstrated a DHT-dependent
increase in the amount of AR (Fig. 2C, bottom panel, West-
ern blot AR lane 2 versus 4). The approximately twofold
increase in the abundance of AR by Western blotting sug-
gests that the increase in the amount of acetylated AR that
was immunoprecipitated in the presence of DHT may be
due to an increase in both absolute and relative amounts of
AR in the presence of DHT. The preclearing by immuno-
precipitation with p53 was saturating (Fig. 2D, lanes 1, 3,
and 5 versus 2, 4, and 6).
The addition of DHT with TSA also increased the amount of
AR coprecipitated with the anti-acetyl-lysine motif antibody
(Fig. 2E, lane 2 versus 4). Again, the preclearing with p53 was
saturating (Fig. 2F, lanes 1 and 3 versus 2 and 4). Together,
these studies demonstrate that the AR is acetylated in vivo and
that, in prostate cancer cells, the addition of DHT or TSA
increases the amount of acetylated AR.
AR coactivation is acetylation site dependent. Acetylated
lysine residues form a docking site recognized by bromodo-
main-containing proteins (21, 24, 33). The coactivator proteins
TABLE 1. Activation by AR coactivatorsa
aThe MMTV-Luc reporter was transfected into DU145 cells with the expres-
sion vector for hAR and the AR coactivators as detailed in Materials and
Methods, and cells were treated with DHT (10?7M) for 24 h. Data are means
? standard errors of the means compared with the control empty expression
VOL. 23, 2003ANDROGEN RECEPTOR ACETYLATION 8567
p300/CBP contain a bromodomain, and an 18-amino-acid
polypeptide containing the AR acetylation site was sufficient
for binding and acetylation by either p300 (Fig. 1A, B) or
p300/CBP-associated factor (P/CAF) (7). The binding of p300
to wild-type AR and the AR mutants was compared by immu-
noprecipitation-Western blot analysis in 293T cells transfected
with expression vectors for p300 and either wild-type AR or
the AR mutants. After 24 h of DHT treatment, the cell lysates
FIG. 4. Reduced N-CoR and HDAC1 binding of AR acetylation site mutants. (A) 293T cells transfected with the ARs were subjected to either
direct Western blotting or AR immunoprecipitation and Western blotting for HDAC1 and AR. GDI served as the loading control. (B) The binding
of HDAC1 to the AR is shown as percent binding for multiple separate transfections. (C) DU145 cells were cotransfected with the MMTV-Luc
reporter and the expression vector for the wild-type AR, mutant AR, or empty expression vector cassette (pcDNA3) and treated with TSA for 24 h.
(D) 293T cells were transfected with the ARs and Flag-N-CoR, treated with DHT (10?7M), and the cellular extracts were subjected to Western
blotting or immunoprecipitation (anti-M2-Flag antibody for N-CoR) and subsequent AR Western blotting. (E) AR immunoprecipitation of cells
transfected with AR acetylation site mutants and Smad3 with sequential Western blotting for Smad3. (F) DU145 Cells were transfected with the
MMTV-Luc reporter and expression vectors for AR, Smad3, or Smad3?C. The DHT-induced change in MMTV-Luc activity is shown as the mean
? standard error of the mean.
8568 FU ET AL.MOL. CELL. BIOL.
were subjected to either Western blotting (Fig. 3A) or immu-
noprecipitation-Western blotting (Fig. 3B).
The wild-type AR and AR mutants were expressed to sim-
ilar levels, and the relative abundance of p300 was similar
between samples in transfected cells (Fig. 3A). In three sepa-
amounts of ARK630Qor ARK630Twere associated with p300
compared with wild-type AR (Fig. 3C). Conversely, a tiny
nonpolar substitution, ARK630A, showed reduced binding of
p300 (Fig. 3D), consistent with a role for an acetylation-in-
duced increase in polarity and/or hydrophobicity in docking to
bromodomain coactivators. We examined whether direct acet-
ylation of recombinant AR could alter the affinity for p300 in
pull-down experiments. In the presence of p300 and acetyl-
coenzyme A, the AR fusion protein 624-676 is efficiently acety-
lated (7). Under the same conditions, a semiquantitative GST
pull-down experiment was conducted in the presence and ab-
sence of acetyl-coenzyme A (Fig. 3E). p300 bound the AR
fragment in the pull-down, and the association of p300 with the
unacetylated AR was disrupted above 0.6 M salt. p300 binding
with acetylated AR held up under more stringent salt washing
conditions, suggesting that AR acetylation enhances p300
binding (Fig. 3E, lane 2 versus 5).
To consider the alternative possibility, that in the presence
of AR, acetyl-coenzyme A may enhance acetylation of p300
and thereby regulate AR binding and function, we conducted
in vitro acetylation experiments with the AR fusion protein
and assessed the autoacetylation of p300 (Fig. 3F). The addi-
tion of AR peptide did not increase p300 autoacetylation,
suggesting that augmentation of p300 acetylation did not in-
dependently contribute to increasing the binding of p300 to the
AR. p300 overexpression enhanced activity of both the unli-
ganded (Fig. 3G) and liganded wild-type AR (Fig. 3H). The
AR acetylation site mutants were activated relatively more by
p300 either in the absence (9- to 10-fold) (Fig. 3G) or in the
presence (4.5-fold) of ligand (Fig. 3H). Since p300 may serve as
a platform to recruit AR coactivators, we examined the role of
the AR acetylation site mimic in regulating AR coactivation by
ARA55, ARA70, SRC1a, TIP60, and Ubc-9 (34). Each of the
AR coactivators augmented activity of the liganded AR mu-
tants more than wild-type AR (Table 1).
AR acetylation site regulates corepressor binding and func-
tion. The repression function of several unliganded NR, me-
diated by N-CoR/SMRT (nuclear receptor corepressor/silenc-
ing mediator for retinoid and thyroid hormone receptors),
involves recruitment of HDACs, TBL1, or basal transcriptional
components (TFIIB, TAFII32, TAFI70) (5, 8, 11, 18). Further-
more, the AR binds to HDAC1 in the liver in vivo (5, 8, 11, 18).
AR-deficient 293T cells transfected with wild-type AR and the
AR mutants showed similar levels of AR and HDAC1 expres-
sion by Western blotting (Fig. 4A, lanes 1 to 3). Saturating AR
immunoprecipitation with subsequent Western blotting for the
AR and HDAC1 showed that HDAC1 binding to the AR
mutants was reduced by 60 to 85% (Fig. 4B). The specific
HDAC inhibitor TSA induced wild-type AR activity with
MMTV-Luc (1.8-fold at 5 nM and 5.2-fold at 10 nM) (Fig. 4C)
consistent with the binding of HDAC1 to the AR. In contrast,
the AR acetylation site mutants were not activated by TSA at
5 nM and conveyed reduced TSA induction at 10 nM (Fig. 4C)
(P ? 0.05, n ? 9).
blot assays increased
The N-CoR N-terminal repression domain contacts NR and
interacts with HDAC complexes (19). Western blot analysis of
AR-deficient 293T cells transfected with AR and Flag-tagged
N-CoR revealed similar levels of N-CoR and AR proteins (Fig.
4D, lanes 1, 2, and 3). Anti-Flag antibody immunoprecipitation
showed similar amounts of N-CoR (Fig. 4D, lanes 4, 5, and 6)
with reduced levels of AR mutants in the N-CoR immunopre-
cipitate (Fig. 4D, lane 4 versus 5 and 6). AR immunoprecipi-
tation with sequential Western blotting for AR or Smad3 dem-
onstrated similar levels of AR but reduced Smad3 binding to
the AR acetylation site mutant (Fig. 4E). Smad3, a component
of the N-CoR complex (14), inhibited liganded wild-type AR
function, and deletion of the Smad3 carboxyl-terminal AR-
binding region, abrogated repression (Fig. 4F). The AR acet-
ylation site mutant remained three- to fivefold more active
than wild-type AR. Together these studies suggest that the
reduced binding of N-CoR/HDAC/Smad3 to the AR mutants
correlated with reduced TSA responsiveness.
AR acetylation site mimic regulates prostate cellular growth
and apoptosis in vivo. To determine the biological properties
regulated by AR acetylation, stable prostate cancer cell lines
(DU145) expressing either wild-type AR or the AR acetylation
site mimic or control vector pcDNA3 were examined for effects
on cellular growth and apoptosis. In view of the reduced bind-
ing of HDAC1 to the AR acetylation mutants and reduced
N-CoR binding in the presence of DHT, we examined DU145
stable cell lines expressing either wild-type AR, ARK630Qor
ARK630Tfor cellular growth and effects of HDAC inhibition.
The indirect measure of cellular proliferation, the MTT assay,
was used first to assess the effects of the HDAC inhibitor TSA
(Fig. 5A) and SAHA (Fig. 5B). The cellular proliferation rate
of the AR acetylation mutant stable cell lines was increased in
the presence of DHT compared to the AR wild type. The
addition of TSA reduced MTT activity by 20% in the wild-type
AR cells. The AR acetylation mutants demonstrated increased
MTT activity in the presence of TSA compared to the AR wild
type. Cellular proliferation was also increased in the AR acet-
ylation mutant cell lines compared to the wild-type AR line.
The cellular proliferation was relatively resistant to inhibition
by the HDAC inhibitor SAHA.
Colony formation was next assessed in DU145 cells express-
ing either the empty vector, wild-type AR, or the AR acetyla-
tion mutants. The size and number of soft agar colonies of
ARK630Tand ARK630Qwere substantially increased compared
with wild-type AR in either the presence or absence of ligand
(Fig. 5C and D and data not shown). The major growth ad-
vantage in colonies containing greater than a hundred cells was
observed in the absence of ligand, suggesting that a basal-level
function of the AR acetylation mutants may contribute in an
important manner to the size of colonies. The mechanism by
which DHT enhances colony formation of DU145 in the ab-
sence of the AR may involve receptor-independent effects of
DHT through other receptors or intracellular kinases and re-
mains to be determined (22, 25). The AR acetylation site
mutations conferred a growth advantage on stable prostate
cancer cell lines compared with wild-type AR in vivo when
implanted in nude mice, forming tumors approximately 20 days
earlier and achieving a volume twice that of wild-type AR. The
ARK630Tclones formed tumors within 37 days (Fig. 5E and F),
and at 120 days (mean ? 22.5 mm3) and were some 4.5-fold
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8570 FU ET AL.MOL. CELL. BIOL.
larger than the wild-type AR (mean ? 5.2 mm3) (Fig. 5F, n ?
5). Apoptosis by TUNEL staining was reduced three- to four-
fold in tumors harboring the AR acetylation mimic mutants
compared with the wild-type AR-expressing clones (Fig. 5G
and H, n ? 4).
The cellular proliferation rate of the ARK630Q and
ARK630Tstable lines, assessed by cell counting, was also
greater than that of wild-type AR (Fig. 6A). A significant
difference in cell numbers was observed at 5 days, where a
trend was observed at day 4 in the presence of DHT (Fig.
6B). These results are consistent with the doubling time of
DU145 cells (36 h) (44). Apoptosis induced by TRAIL and
cycloheximide was reduced twofold in the ARK630Qand
ARK630Tlines compared with wild-type AR (Fig. 6C). Ex-
periments were conducted on at least three stable lines on
three separate experiments. Mitogen-activated protein ki-
nase kinase kinase 1 (MEKK1) induces prostate cancer cell
apoptosis in an AR-dependent manner (2) (Fig. 6E). In the
presence of DHT and wild-type AR expression, with mor-
phological analysis of GFP-transfected cells as previously
described (20), cellular apoptosis was 15%. MEKK1 in-
creased liganded wild-type AR apoptosis rates from 15% to
30 to 35% (Fig. 6F, lane 3 versus 5), again consistent with
prior studies. MEKK1-induced apoptosis of ARK630Twas,
however, reduced 50% compared with wild-type AR and
was abolished in cells expressing ARK630Q(Fig. 6F, lane 5
versus 9 and 13).
To investigate the mechanism by which AR acetylation mu-
tant may convey enhanced cellular proliferation, we assessed
the effects of the AR mutants on the activity of cell cycle
control gene promoters. Compared with wild-type AR, the AR
acetylation mimic mutants increased activity of the cell cycle
control genes cyclin D1 two- to fourfold and the cyclin E
promoter 1.5-fold (Fig. 6G and H). Activity of the p21Cip1
promoter was regulated equally by expression of either wild-
type AR or an AR mutant, suggesting that a subset of growth
control genes are regulated by the AR acetylation mimic mu-
tants. Western blot analysis of the DU145 stable cell line was
consistent with the reporter data. Cyclin D1 and cyclin E pro-
tein levels were both increased in the AR acetylation mutant
cell lines compared to the AR wild-type cell lines when nor-
malized to the loading control GDI (Fig. 6I). Together, these
studies demonstrate that the AR acetylation mutant stable cell
lines exhibit enhanced basal and DHT-induced cellular prolif-
eration and reduced cellular apoptosis in response to TRAIL
and cycloheximide. The enhanced growth advantage corre-
lated with increased activation of the promoters for the cell-
cycle control genes cyclin D1 and cyclin E.
Phosphorylation of receptors or transcription factors has
been linked to altered contact-independent cellular growth
(20, 23). Herein, transcription factor acetylation is shown to
govern cellular growth in vivo. Prostate cancer stable cells
overexpressing AR acetylation mimic mutants grew faster,
formed larger numbers of colonies and colonies of greater size,
and, when implanted in nude mice, grew faster than the wild-
type AR lines. The AR was acetylated in vivo, and the acety-
lation was regulated by DHT and TSA. The acetylation site
mimic mutants regulated a subset of AR functions.
The AR conveys both ligand-dependent and -independent
(aporeceptor) functions. Previous studies demonstrated that
the AR acetylation site did not affect trans-repression (NF-?B,
Sp1), sumoylation, or global structure assessed by protease
digestion (7). In the current studies, the AR acetylation site
affected aporeceptor function (TRAIL-mediated apoptosis,
HDAC1 binding, cellular proliferation, contact-independent
growth, induction of cell cycle gene promoters), and ligand-
induced activities (ARE trans-activation, N-CoR, p300 bind-
ing). As the AR acetylation mimic mutants conveyed a cellular
proliferate advantage and evaded MEKK1- and TRAIL-medi-
ated apoptosis, it is plausible that both characteristics contrib-
uted to the growth advantage identified in vivo.
Previous studies have shown that the AR is acetylated in
vitro by the HATs p300, P/CAF, and TIP60 (7–10). Several
lines of evidence in the current study suggest that the AR is
acetylated in vivo. Reciprocal immunoprecipitation-Western
blotting was conducted on mouse liver tissues. The immuno-
precipitate with the AR antibody, which contained immunore-
active AR, was cross-reactive with the anti-acetyl-lysine anti-
body at the same mobility as the AR by Western blotting.
Conversely, the anti-acetyl-lysine antibody-immunoprecipi-
tated protein reacted specifically with the AR antibody. Sec-
ond, antibodies generated against an acetylated peptide resem-
bling the acetylated AR motif immunoprecipitated the AR.
Third, the HDAC inhibitor TSA and the AR ligand DHT
induced acetylation of the AR in a prostate cancer cell line.
In the current studies, the reduced binding of N-CoR/
HDAC1 and increased binding of p300 to ARK630Qand
ARK630Tsuggests that Lys630contributes to the molecular sur-
face that interacts with both coactivators and corepressors.
Acetylation of lysine residues through neutralizing the
residue’s positive charge and increasing its polarity may alter
intra- and intermolecular interactions. The enhanced ligand-
dependent transcriptional activity of the ARK630Q and
ARK630Tmutants, together with enhanced p300 binding and
coactivator induction, suggest that neutralization of the
FIG. 5. AR acetylation mutants convey contact-independent growth. (A and B) MTT assay of DU145 stable cell lines expressing either
pcDNA3, wild-type AR, or AR acetylation site mutants. Equal numbers of cells were seeded into 96-well plates, treated with either DHT, TSA,
or SAHA for 24 h, and the MTT assay was conducted, measuring absorbance at 570 nm. (C and D) DU145 cells stably expressing wild-type AR
or AR acetylation site mutants were seeded in soft agar. Phase contrast image of the colonies from a representative experiment is shown
(magnification, ?100). Colony numbers and size (percentage of colonies with ?100 cells) are shown at day 14. (E and F) Nude mice were
implanted with 106cells of stable lines expressing either wild-type AR or AR acetylation site mutants. The mean volume of DU145 tumors grown
in nude mice were shown at each time point. (G and H) Apoptosis in implanted tumors was assessed by TUNEL staining for wild-type AR,
ARK630Q, and ARK630T(n ? 4).
VOL. 23, 2003 ANDROGEN RECEPTOR ACETYLATION8571
ARK630charge, or changes in size or shape at the side chain,
plays a key role in coactivator recruitment and trans-activation.
The finding that the coactivator and corepressor surfaces of
NR overlap substantially (19, 32) is compatible with a dynamic
model in which enzymatic modifications of the nuclear recep-
tors by acetylation coordinates sequential disengagement of
corepressors followed by coactivator binding (29). Our obser-
vations contribute to a more general model of transcription
factor regulation in which acetylation contributes to corepres-
sor disengagement with sequential coactivator recruitment
Several lines of evidence suggest that the alterations in co-
activator binding of the AR acetylation mimic are specific.
First, we show that acetylation of the AR physically enhances
association with p300. Second, the gain-of-function acetylation
mimic mutants showed enhanced binding to p300, whereas
substitution of a residue that does not mimic acetylation (ala-
nine) did not enhance p300 binding. Third, the gain-of-func-
tion acetylation mimic mutants showed reduced binding to
HDAC1 and N-CoR, whereas the alanine substitution mutant
showed enhanced corepressor (N-CoR) binding. Fourth, the
two acetylation mimic point mutants (ARK630Qand ARK630T)
had gain of function in transcriptional reporter assays, whereas
the acetylation “dead” mutants, alanine (ARK630A) and argi-
nine (ARK630R) substitutions, showed loss of ligand-induced
trans-activation. Fifth, our previous studies showed that point
mutation of the acetylated lysine residues abrogated immuno-
reactivity with the anti-acetyl-lysine antibody and that the gross
conformation of the AR mutated at the lysine residue is unal-
tered in protease sensitivity assays. Sixth, acetylation mutations
do not affect sumoylation of the AR or AR trans-repression of
NF-?B and Sp1 signaling (7, 8), demonstrating that the AR
mutants maintain several normal functions of the receptor and
that the acetylation site affects only a subset of AR activities.
Finally, the acetylation mimic AR mutants showed selective
enhancement of trans-activation of a subset of growth control
FIG. 6. AR acetylation site governs cellular proliferation and MEKK1-dependent apoptosis. (A and B) Enhanced cellular proliferation rate of
AR acetylation site mutants. A total of 2 ? 104DU145 cells stably transfected with the expression vector for either wild-type AR, AR mutants,
or control vector pcDNA3 were seeded and treated either with vehicle (A) or DHT (10?7M) (B). The representative results of three independent
experiments are shown. (C) AR acetylation site prostate cancer cell lines are resistant to TRAIL-induced apoptosis. Cell survival rates of DU145
stable cell lines exposed to TRAIL (10 ng/ml) and cycloheximide (CHX; 10 ?g/ml) are shown compared with untreated cells (100%). (D) Phase
contrast of cell lines is shown. (E, F). AR acetylation site mutants evade MEKK-1-mediated apoptosis. DU145 cells were transfected with MEKK1,
AR, and pCMV-GFP as indicated and treated with either vehicle or DHT (10 nM) for 24 h. The morphology of the transfected DU145 cells is
shown in phase contrast. (E) White arrows indicate GFP-positive cells, and yellow arrows indicate GFP-positive cells showing chromatin
condensation. (F) The graph represents independent experiments in which 200 green fluorescent cells were counted and scored for cytoplasmic
blebbing and chromatin condensation; ?, P ? 0.01 for the effect of MEKK1 on liganded AR-induced apoptosis. (G and H) AR acetylation site
mutants alter regulation of cell cycle control genes. Reporter assays showing regulation of cell cycle control promoters by wild-type AR or AR
acetylation site mutants in DU145 cells treated with vehicle (G) or DHT (H). (I) Western blot analysis of DU145 stable cell lines stably expressing
wild-type AR and AR acetylation mutants. Cells were starved for 24 h and harvested 6 h after treatment (10% charcoal-stripped fetal bovine serum
plus 100 nM DHT). GDI served as a protein-loading control.
8572 FU ET AL.MOL. CELL. BIOL.
gene promoters (cyclin D1, cyclin E, and cyclin A). Thus, each
of these selective gains of function of the AR acetylation mimic
mutants are the opposite to the function of the acetylation-
dead AR mutants.
Our studies demonstrate that select subsets of functions of
the AR are regulated by the acetylation site of the AR, both
basally and ligand induced, that together contribute to aber-
rant growth control. The AR conveys several distinct functions
in the absence of ligand (aporeceptor) and in the presence of
ligand. The unliganded functions of the AR include trans-
activation of synthetic AREs, trans-activation of native pro-
moters, trans-repression (NF-?B, Sp1), and regulation of apo-
ptosis and growth. The current studies show a role for the AR
acetylation site in the absence of ligand in regulation of cell
cycle control genes, reduced apoptosis in the presence of
TRAIL/cycloheximide, and altered binding of HDAC1. In the
presence of ligand, the AR acetylation site mutants showed
increased trans-activation of synthetic and natural AREs, ei-
ther alone or in the presence of coactivators. The acetylation
sites do not appear to affect the trans-repression function of
synthetic reporters or sumoylation of the AR (7, 8).
In the current studies, mutation of the AR acetylation site
reduced apoptosis in response to TRAIL and cycloheximide or
MEKK1. The role of the AR in basal and ligand-induced
apoptosis is controversial, some investigators considering the
AR an inducer of apoptosis (16, 26) and others as a prosurvival
factor (9, 43, 46). Here we considered that the AR acetylation
mutants conveyed a “gain of function” of enhanced survival.
The acetylation mimic mutant showed a growth advantage in
the absence of ligand coinciding with the enhanced trans-acti-
vation of the native promoters for cell-cycle and growth control
genes (cyclin D1, cyclin E and cyclin A) in the absence of
ligand, suggesting that the altered regulation of these genes
may contribute to the increased cellular growth.
The tumor suppressor p53 and the related p73 protein are
acetylated (35), and substitution of the acetylated residues of
p73 with charged arginine residues also blocked both apoptotic
and p300-dependent trans-activation functions (6). Our studies
demonstrate that charge changes at the AR acetylated lysine
residue alter the type of coactivator/corepressor complexes
recruited to the AR. Neutral polar substitutions (ARK630Q,
ARK630T) enhanced p300 binding and reduced N-CoR/HDAC/
VOL. 23, 2003 ANDROGEN RECEPTOR ACETYLATION8573
Smad3 corepressor binding, whereas unacetylatable inactivat-
ing mutants (ARK630R, ARK630A) reduced p300 binding and
enhanced corepressor binding (8). As all of the ARK630muta-
tions and the p73 mutants were resistant to the induction of
apoptosis and by design are incapable of receiving an acetyl
group from acetyl-coenzyme A at the native lysine residue, it is
possible that the acetyl group, once transferred to the lysine
acceptor, forms a platform involved in subsequent apoptosis.
The effectiveness of androgen ablation therapy in reducing
prostate cancer cell growth suggests a key role for the liganded
AR in aberrant prostate cell growth (1). Therapeutic resistance
to the androgen antagonist hydroxyflutamide contributes to
morbidity and mortality in human prostate cancer. The iden-
tification of acetylation in the current studies as a key post-
translational modification regulating AR growth control sug-
gests that ARK630may form an ideal target for novel tumor
We thank Paul A. Marks, D. Chadee, R. Evans, E. Kalkhoven,
Victoria M. Richon, J. M. Kyriakis, Y. Nakatani, B. O’Malley, and N.
Schreiber-Agus for plasmids and helpful discussions.
This work was supported by grants from NIH (R01CA70896,
R01CA75503, R01CA86072), the Pfeiffer Foundation, the Susan
R21DK065220-01 (NIDDK) (to M.F.), R03 AG2033 (to C.A.), R01-
CA65647 (to S.P.B.), and R01 CA83979 (to M.L.A.). Work conducted
at the Lombardi Cancer Center was supported by Comprehensive
Cancer Center Core National Institute of Health grant P30 CA51008-
1. Abate-Shen, C., and M. M. Shen. 2000. Molecular genetics of prostate
cancer. Genes Dev. 14:2410–2434.
2. Abreu-Martin, M. T., A. Chari, A. A. Palladino, N. A. Craft, and C. L.
Sawyers. 1999. Mitogen-activated protein kinase kinase kinase 1 activates
androgen receptor-dependent transcription and apoptosis in prostate cancer.
Mol. Cell. Biol. 19:5143–5154.
3. Alland, L., R. Muhle, H. J. Hou, J. Potes, L. Chin, N. Schreiber-Agus, and
R. A. DePinho. 1997. Role for N-CoR and histone deacetylase in Sin3-
mediated transcriptional repression. Nature 387:49–55.
4. Chen, D., H. Ma, H. Hong, S. S. Koh, S. M. Huang, B. T. Schurter, D. W.
Aswad, and M. R. Stallcup. 1999. Regulation of transcription by a protein
methyltransferase. Science 284:2174–2177.
5. Chen, J. D., and R. M. Evans. 1995. A transcriptional co-repressor that
interacts with nuclear hormone receptors. Nature 377:454–457.
6. Costanzo, A., P. Merlo, N. Pediconi, M. Fulco, V. Sartorelli, P. A. Cole, G.
Fontemaggi, M. Fanciulli, L. Schiltz, G. Blandino, C. Balsano, and M.
Levrero. 2002. DNA damage-dependent acetylation of p73 dictates the se-
lective activation of apoptotic target genes. Mol. Cell. 9:175–186.
7. Fu, M., C. Wang, A. T. Reutens, J. Wang, R. H. Angeletti, L. Siconolfi-Baez,
V. Ogryzko, M. L. Avantaggiati, and R. G. Pestell. 2000. p300 and p300/
cAMP-response element-binding protein-associated factor acetylate the an-
drogen receptor at sites governing hormone-dependent transactivation.
J. Biol. Chem. 275:20853–20860.
8. Fu, M., C. Wang, J. Wang, X. Zhang, T. Sakamaki, Y. G. Yeung, C. Chang,
T. Hopp, S. A. Fuqua, E. Jaffray, R. T. Hay, J. J. Palvimo, O. A. Janne, and
R. G. Pestell. 2002. Androgen receptor acetylation governs trans activation
and MEKK1-induced apoptosis without affecting in vitro sumoylation and
trans-repression function. Mol. Cell. Biol. 22:3373–3388.
9. Gao, J., and J. T. Isaacs. 1998. Development of an androgen receptor-null
model for identifying the initiation site for androgen stimulation of prolif-
eration and suppression of programmed (apoptotic) death of PC-82 human
prostate cancer cells. Cancer Res. 58:3299–3306.
10. Gaughan, L., I. R. Logan, S. Cook, D. E. Neal, and C. N. Robson. 2002. Tip60
and Histone Deacetylase 1 Regulate Androgen Receptor Activity through
Changes to the Acetylation Status of the Receptor. J. Biol. Chem. 277:
11. Glass, C. K., and M. G. Rosenfeld. 2000. The coregulator exchange in
transcriptional functions of nuclear receptors. Genes Dev. 14:121–141.
12. Harrod, R., J. Nacsa, C. Van Lint, J. Hansen, T. Karpova, J. McNally, and
G. Franchini. 2002. Human immunodeficiency virus type-1 Tat/Co-activator
acetyltransferase interactions inhibit p53K320-acetylation and p53-respon-
sive transcription. J. Biol. Chem. 278:12310–12318.
13. Hayes, S., M. Zarnegar, M. Sharma, F. Yang, D. M. Peehl, P. ten Dijke, and
Z. Sun. 2001. SMAD3 represses androgen receptor-mediated transcription.
Cancer Res. 61:2112–2118.
14. Hayes, S. A., M. Zarnegar, M. Sharma, F. Yang, D. M. Peehl, P. ten Dijke,
and Z. Sun. 2001. SMAD3 represses androgen receptor-mediated transcrip-
tion. Cancer Res. 61:2112–2118.
15. Heinzel, T., R. M. Lavinsky, T. M. Mullen, M. Soderstrom, C. D. Laherty, J.
Torchia, W. M. Yang, G. Brard, S. D. Ngo, J. R. Davie, E. Seto, R. N.
Eisenman, D. W. Rose, C. K. Glass, and M. G. Rosenfeld. 1997. A complex
containing N-CoR, mSin3 and histone deacetylase mediates transcriptional
repression. Nature 387:43–48.
16. Heisler, L. E., A. Evangelou, A. M. Lew, J. Trachtenberg, H. P. Elsholtz, and
T. J. Brown. 1997. Androgen-dependent cell cycle arrest and apoptotic death
in PC-3 prostatic cell cultures expressing a full-length human androgen
receptor. Mol. Cell. Endocrinol. 126:59–73.
17. Higashimoto, Y., S. Saito, X. H. Tong, A. Hong, K. Sakaguchi, E. Appella,
and C. W. Anderson. 2000. Human p53 is phosphorylated on serines 6 and
9 in response to DNA damage-inducing agents. J. Biol. Chem. 275:23199–
18. Horlein, A. J., A. M. Naar, T. Heinzel, J. Torchia, B. Gloss, R. Kurokawa, A.
Ryan, Y. Kamei, M. Soderstrom, C. K. Glass, et al. 1995. Ligand-indepen-
dent repression by the thyroid hormone receptor mediated by a nuclear
receptor co-repressor. Nature 377:397–404.
19. Hu, X., Y. Li, and M. A. Lazar. 2001. Determinants of CoRNR-dependent
repression complex assembly on nuclear hormone receptors. Mol. Cell. Biol.
20. Hunter, T. 1997. Oncoprotein networks. Cell. 88:333–346.
21. Jacobson, R. H., A. G. Ladurner, D. S. King, and R. Tjian. 2000. Structure
and function of a human TAFII250 double bromodomain module. Science
22. Janssen, T., R. Kiss, R. Dedecker, M. Petein, J. L. Pasteels, and C. Schul-
man. 1995. Influence of dihydrotestosterone, epidermal growth factor, and
basic fibroblast growth factor on the cell kinetics of the PC3, DU145, and
LNCaP prostatic cancer cell lines: relationship with DNA ploidy level. Pros-
23. Karin, M., and T. Hunter. 1995. Transcriptional control by protein phos-
phorylation: signal transmission from the cell surface to the nucleus. Curr.
24. Kouzarides, T. 2000. Acetylation: a regulatory modification to rival phos-
phorylation. EMBO J. 19:1176–1179.
25. Lee, Y. F., W. J. Lin, J. Huang, E. M. Messing, F. L. Chan, G. Wilding, and
C. Chang. 2002. Activation of mitogen-activated protein kinase pathway by
the antiandrogen hydroxyflutamide in androgen receptor-negative prostate
cancer cells. Cancer Res. 62:6039–6044.
26. Lin, H. K., S. Yeh, H. Y. Kang, and C. Chang. 2001. Akt suppresses andro-
gen-induced apoptosis by phosphorylating and inhibiting androgen receptor.
Proc. Natl. Acad. Sci. 98:7200–7205.
27. Liu, L., D. M. Scolnick, R. C. Trievel, H. B. Zhang, R. Marmorstein, T. D.
Halazonetis, and S. L. Berger. 1999. p53 sites acetylated in vitro by PCAF
and p300 are acetylated in vivo in response to DNA damage. Mol. Cell. Biol.
28. Mauser, A., S. Saito, E. Appella, C. W. Anderson, W. T. Seaman, and S.
Kenney. 2002. The Epstein-Barr virus immediate-early protein BZLF1 reg-
ulates p53 function through multiple mechanisms. J. Virol. 76:12503–12512.
29. McKenna, N. J., R. B. Lanz, and B. W. O’Malley. 1999. Nuclear receptor
coregulators: cellular and molecular biology. Endocrinol. Rev. 20:321–344.
30. Nagy, L., H. Y. Kao, D. Chakravarti, R. J. Lin, C. A. Hassig, D. E. Ayer, S. L.
Schreiber, and R. M. Evans. 1997. Nuclear receptor repression mediated by
a complex containing SMRT, mSin3A and histone deacetylase. Cell. 89:373–
31. Ogryzko, V. V., R. L. Schiltz, V. Russanova, B. H. Howard, and Y. Nakatani.
1996. The transcriptional coactivators p300 and CBP are histone acetyltrans-
ferases. Cell. 87:953–959.
32. Perissi, V., L. M. Staszewski, E. M. McInerney, R. Kurokawa, A. Krones,
D. W. Rose, M. H. Lambert, M. V. Milburn, C. K. Glass, and M. G.
Rosenfeld. 1999. Molecular determinants of nuclear receptor-corepressor
interaction. Genes Dev. 13:3198–3208.
33. Polesskaya, A., I. Naguibneva, A. Duquet, E. Bengal, P. Robin, and A.
Harel-Bellan. 2001. Interaction between acetylated MyoD and the bromo-
domain of CBP and/or p300. Mol. Cell. Biol. 21:5312–5320.
34. Poukka, H., P. Aarnisalo, U. Karvonen, J. J. Palvimo, and O. A. Janne. 1999.
Ubc9 interacts with the androgen receptor and activates receptor-dependent
transcription. J. Biol. Chem. 274:19441–19446.
35. Prives, C., and J. L. Manley. 2001. Why is p53 acetylated? Cell. 107:815–818.
36. Reutens, A. T., M. Fu, C. Wang, C. Albanese, M. J. McPhaul, Z. Sun, S. P.
Balk, O. A. Janne, J. J. Palvimo, and R. G. Pestell. 2001. Cyclin D1 binds the
androgen receptor and regulates hormone-dependent signaling in a p300/
CBP-associated factor (P/CAF)-dependent manner. Mol. Endocrinol. 15:
8574FU ET AL.MOL. CELL. BIOL.
37. Sterner, D. E., and S. L. Berger. 2000. Acetylation of histones and transcrip-
tion-related factors. Microbiol. Mol. Biol. Rev. 64:435–459.
38. Struhl, K. 1998. Histone acetylation and transcriptional regulatory mecha-
nisms. Genes Dev. 12:599–606.
39. Tan, J., Y. Sharief, K. G. Hamil, C. W. Gregory, D. Y. Zang, M. Sar, P. H.
Gumerlock, R. W. DeVere White, T. G. Pretlow, S. E. Harris, E. M. Wilson, J. L.
Mohler, and F. S. French. 1997. Dehydroepiandrosterone activates mutant
androgen receptors expressed in the androgen-dependent human prostate can-
cer xenograft CWR22 and LNCaP cells. Mol. Endocrinol. 11:450–459.
40. Taplin, M. E., G. J. Bubley, Y. J. Ko, E. J. Small, M. Upton, B. Rajeshkumar,
and S. P. Balk. 1999. Selection for androgen receptor mutations in prostate
cancers treated with androgen antagonist. Cancer Res. 59:2511–2515.
41. Taplin, M. E., G. J. Bubley, T. D. Shuster, M. E. Frantz, A. E. Spooner, G. K.
Ogata, H. N. Keer, and S. P. Balk. 1995. Mutation of the androgen-receptor
gene in metastatic androgen-independent prostate cancer. N. Engl. J. Med.
42. Torchia, J., C. Glass, and M. G. Rosenfeld. 1998. Co-activators and co-
repressors in the integration of transcriptional responses. Curr. Opin. Cell
43. Vendola, K. A., J. Zhou, O. O. Adesanya, S. J. Weil, and C. A. Bondy. 1998.
Androgens stimulate early stages of follicular growth in the primate ovary.
J. Clin. Investig. 101:2622–2629.
44. Vilenchik, M., A. J. Raffo, L. Benimetskay, D. Shames, and C. A. Stein. 2002.
Antisense RNA down-regulation of bcl-xL expression in prostate cancer cells
leads to diminished rates of cellular proliferation and resistance to cytotoxic
chemotherapeutic agents. Cancer Res. 62:2175–2183.
45. Wang, C., M. Fu, S. Mani, S. Wadler, A. M. Senderowicz, and R. G. Pestell.
2001. Histone acetylation and the cell-cycle in cancer. Front. Biosci. 6:D610–
46. Ye, D., J. Mendelsohn, and Z. Fan. 1999. Androgen and epidermal growth
factor down-regulate cyclin-dependent kinase inhibitor p27Kip1 and co-
stimulate proliferation of MDA PCa 2a and MDA PCa 2b prostate cancer
cells. Clin. Cancer Res. 5:2171–2177.
47. Yuan, S., J. Trachtenberg, G. B. Mills, T. J. Brown, F. Xu, and A. Keating.
1993. Androgen-induced inhibition of cell proliferation in an androgen-
insensitive prostate cancer cell line (PC-3) transfected with a human andro-
gen receptor complementary DNA. Cancer Res. 53:1304–1311.
VOL. 23, 2003 ANDROGEN RECEPTOR ACETYLATION 8575