[CANCER RESEARCH 59, 2297–2301, May 15, 1999]
Advances in Brief
D-Type Cyclins Complex with the Androgen Receptor and Inhibit Its
Transcriptional Transactivation Ability1
Karen E. Knudsen, Webster K. Cavenee, and Karen C. Arden2
Ludwig Institute for Cancer Research [K. E. K., W. K. C., K. C. A.], Department of Medicine [W. K. C., K. C. A.], Center for Molecular Genetics [W. K. C.], and Cancer Center
[W. K. C.], University of California at San Diego, La Jolla, California 92093-0660; and University of Cincinnati School of Medicine, Department of Cell Biology, Cincinnati,
Ohio 45267-0521 [K. E. K.]
Abstract
D-type cyclins regulate distinct cellular processes, such as mitotic cell
cycle control, differentiation, and transcription. We have previously
shown that the D-type cyclins are critical for the androgen-dependent
proliferation of prostate cells. Here, we sought to determine whether
cyclin D1 directly influences the transactivation potential of the androgen
receptor, a transcription factor that strongly influences androgen-dependent proliferation. We found that ligand-mediated transcriptional activation of a physiological target, prostate-specific antigen, by the androgen
receptor was inhibited by cyclins D1 and D3. The ability of D-type cyclins
to inhibit androgen receptor transactivation was not shared with other
cyclins, and cyclin D1 was as effective as dominant negative mutants of the
androgen receptor in inhibiting transactivation. This function of cyclin D1
was independent of its role in cell cycle progression and is likely elicited
through its ability to form a specific complex with the androgen receptor.
These data underscore the various mechanisms through which the androgen receptor is regulated and also point to a negative feedback role for
cyclin D1 in controlling androgen-dependent growth.
Introduction
Multiple cellular functions have been proposed for the D-type
cyclins, including control of cell cycle regulation, differentiation, and
transcriptional regulation (1– 4). Of these, the role of D-type cyclins in
cell cycle control is the best characterized. The D-type cyclins (cyclins
D1, D2, and D3) bind and activate the CDKs3 CDK4 and CDK6 (1).
In turn, active cyclin D/CDK4(6) complexes phosphorylate the retinoblastoma tumor suppressor protein (RB). Overt activation of cyclin
D1 and/or CDK4 is observed in many tumor cells (1, 5) and contributes to their uncontrolled proliferation. Similarly, overexpression of
cyclin D1 in normal cells accelerates the G1 phase of the cell cycle (6).
Recent evidence also points to a direct role for D-type cyclins in
transcriptional activation and repression. For example, it has been shown
that cyclin D1 inhibits transcriptional activation by the DMP1 transcription factor (4). Conversely, a multitude of evidence now shows that cyclin
D1 binds the estrogen receptor and activates its transactivation potential
in a ligand-independent manner (2, 3). In both cases, the influence of
cyclin D1 on transcription has been shown to occur independently of its
role in cell cycle progression, thus defining a novel function for D-type
cyclins as transcriptional regulators.
Prostatic epithelial cells are dependent on specific steroidal hormones for proliferation, and this dependency is commonly lost in
prostatic adenocarcinoma cells (7, 8). Growth of normal prostate cells
is dependent on the presence of androgen, which elicits its biological
activity via the androgen receptor, a member of the nuclear steroid
hormone receptor family (9). DHT is a high-affinity ligand for the
androgen receptor, and upon DHT binding, the transcriptional transactivation potential of the androgen receptor is activated (9). Upon
activation, the androgen receptor stimulates the transcription of a
number of target genes. Although many of these target genes have yet
to be identified, the best characterized to date is that encoding PSA
(9). PSA expression correlates well with active androgen receptors
and the growth of prostate cells and, as such, is used clinically as a
measure of prostate growth (10). We have previously shown that
D-type cyclins play a key regulatory role in the androgen-dependent
proliferation of prostate cells (11). To test whether D-type cyclins
influence the transactivation potential of the androgen receptor, we
investigated the effects of D-type cyclins on the androgen receptor
using the PSA promoter as a reporter of androgen receptor function.
Surprisingly, we found that D-type cyclins repress activation of androgen receptor-mediated transcription and do so in a direct, cell
cycle-independent manner. These findings may explain the low frequency of cyclin D1 amplification in prostatic adenocarcinomas.
Materials and Methods
Cell Culture and Conditions. Twenty-four to 48 h prior to transfection,
CV1 and SAOS-2 cells were cultured in a 5% CO2 incubator in phenol red-free
DMEM supplemented with 10% charcoal dextran-treated fetal bovine serum
(CDT; Hyclone Laboratories, Logan, UT), 2 mM L-glutamine, and 100 units/ml
penicillin/streptomycin (Mediatech, Herndon, VA).
Transcriptional Activation Assays. CV1 and SAOS-2 cells were transfected with the indicated plasmids using the N,N-bis(2-hydroxyethyl)-2amino-ethanesulfonic acid/calcium phosphate method (12) in 60-mm dishes
using 8 mg of total DNA per transfection. Approximately 16 h posttransfection,
cells were washed with PBS and replaced in phenol red-free DMEM-10%
charcoal dextran-treated fetal bovine serum with or without 0.1 nM R1881
(DuPont/NEN, Boston, MA), a DHT analogue. Approximately 30 h later, cells
were harvested and luciferase activity was monitored using commercially
available reagents (Promega, Madison, WI). b-Galactosidase activity was also
monitored as an internal control for transfection efficiency.
Plasmids. The CMV-b-galactosidase, CMV-CDK4, and CMV-cyclin A
constructs were gifts of Dr. Jean Wang (University of California at San Diego,
La Jolla, CA). The pAR0 human androgen receptor construct was a gift of Dr.
A. O. Brinkmann (Erasmus Universiteit, Rotterdam, the Netherlands), and the
pSG5-AR androgen receptor expression construct was a gift of Dr. Chawnshang Chang (University of Rochester, Rochester, NY). The PSA61LUC reReceived 1/28/99; accepted 3/30/99.
porter construct, which contains 6.1 kb of the human PSA promoter, was a gift
The costs of publication of this article were defrayed in part by the payment of page
of Dr. Kitty Cleutjens (Erasmus Universiteit; Ref. 13). The dominant negative
charges. This article must therefore be hereby marked advertisement in accordance with
androgen receptor constructs pSG5-rARD46 – 408 and pSG5-rARD38 –296
18 U.S.C. Section 1734 solely to indicate this fact.
1
were provided by J. Palvimo and O. Janne (University of Helsinki, Helsinki,
K. E. K. is supported by a National Research Service Award from NIH (Grant
Finland; Ref. 14). The RSV-cyclin D1 and RSV-cyclin D3 constructs were
CA82034).
2
To whom requests for reprints should be addressed, at Ludwig Institute for Cancer
kindly provided by Dr. C. Sherr (St. Jude Children’s Research Hospital,
Research, 9500 Gilman Drive, La Jolla, CA 92093-0660. Phone: (619) 534-7812; Fax:
Memphis, TN). The pRcCyclin D1-GH and pRcCyclin D1-KE constructs were
(619) 534-7802; E-mail: [email protected].
3
gifts of Dr. Robert Weinberg (Whitehead Institute for Biomedical Research,
The abbreviations used are: CDK, cyclin-dependent kinase; DHT, dihydrotestosterCambridge, MA). The cyclin E expression construct (expressed from a long
one; PSA, prostate-specific antigen; CMV, cytomegalovirus; RSV, Rous sarcoma virus.
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CYCLIN D INHIBITS ANDROGEN RECEPTOR TRANSCRIPTION
terminal repeat) was obtained from Dr. J. Roberts (Fred Hutchinson Cancer
Research Center, Seattle, WA).
Immunoprecipitation and Immunoblots. CV1 cells were cultured in
10-cm dishes and cotransfected with 8 mg of pAR0 and 8 mg of RSV-cyclin D1.
Approximately 48 h posttransfection, cells were harvested and lysed in NETN [20
mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0), and 0.5% NP40]
supplemented with 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml 1,10-phenanthroline, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mM sodium fluoride, 1 mM
sodium vanadate, and 60 mM b-glycerophosphate. Following brief sonication and
clarification, lysates were subjected to immunoprecipitation with antisera against
MDM2 (Santa Cruz Biotechnology, Santa Cruz, CA), the androgen receptor
(Santa Cruz Biotechnology), cyclin E (Santa Cruz Biotechnology), cyclin A (Santa
Cruz Biotechnology), or cyclin D1 (Santa Cruz Biotechnology). Immunoprecipitates were recovered using protein A-Sepharose (Pharmacia) and were subjected to
10.5% SDS-PAGE. For immunoprecipitates from cells transfected with cyclin D1
and the androgen receptor, following transfer to Immobilon (Millipore), the
membrane was split at the Mr 50,000 marker, the top half was probed using the
anti-androgen receptor antibody, and the lower half was probed using the anticyclin D1 antibody. For immunoprecipitates from cells transfected with cyclin A
and the androgen receptor, following transfer to Immobilon (Millipore), samples
were run in duplicate to probe for cyclin A and the androgen receptor. Proteins
were visualized using horseradish peroxidase-conjugated protein A (Bio-Rad) and
enhanced chemiluminescence (Amersham).
Results
Cyclin D1 Represses Androgen Receptor Activity. The D-type
cyclins have been shown to play a key regulatory role in the androgendependent proliferation of prostate cells (11). To determine whether
cyclin D1 affects the transactivation potential of the androgen receptor,
initial reporter assays were performed using CV1 cells. These cells were
chosen as they are nontumorigenic and express no measurable androgen
receptor or androgen receptor activity (data not shown). The androgen
receptor was introduced by transfection into CV1 cells using one of two
wild-type androgen receptor expression constructs, pAR0 or pSG5-AR.
To activate ectopically expressed androgen receptors when appropriate,
we treated transfected cells with R1881, a potent ligand of the androgen
receptor. To monitor androgen receptor activation, the PSA61LUC reporter construct was used. PSA61LUC was constructed using 6.1 kb of
the PSA promoter cloned upstream of luciferase (13). Transcription from
the PSA promoter is activated by ligand-bound androgen receptors in
vivo, and the promoter contains at least three consensus androgen-responsive elements. Activation of PSA61LUC in the presence of R1881 was
measured by normalizing luciferase activity to b-galactosidase activity as
an internal control for transfection efficiency.
In CV1 cells, introduction of PSA61LUC resulted in only low-level
background luciferase activity in the presence or absence of R1881
(Fig. 1A), as was expected, based on our observation that no endogenous androgen receptor can be detected in these cells (data not
shown). These results also confirm that activation of the PSA promoter is specific to introduction of an activated androgen receptor.
Likewise, CV1 cells transfected with the androgen receptor (pAR0 or
pSG5-AR) but maintained in a steroid-free environment also demonstrated only low-level PSA61LUC activity (Fig. 1A). By contrast,
cells transfected with the androgen receptor and empty vector RcRSV,
and treated posttransfection with R1881 demonstrated a significant
(5–10-fold) increase in PSA61LUC activation. Strikingly, we observed that cotransfection of RSV-cyclin D1 at a 3:1 ratio with the
androgen receptor resulted in a dramatic inhibition of PSA61LUC
activation (Fig. 1A). This reduced androgen receptor transactivation
potential was observed with both the pAR0 and pSG5-AR constructs.
Interestingly, cyclin D1 specifically inhibited the ligand dependent
activity of PSA61LUC and not the basal activity.
To verify these observations, titration experiments were performed,
wherein cells were transfected with constant amounts of total plasmid
at 1:0, 1:1, 1:3, or 1:5 ratios of androgen receptor to cyclin D1. As
observed in Fig. 1B, cyclin D-mediated inhibition of androgen receptor
activation could be observed at a 1:1 ratio (2-fold decrease), was more
pronounced at a 1:3 ratio (3-fold decrease), and forced measurable
PSA61LUC activity to below background at a 1:5 ratio.
To determine whether this transcriptional modulation was specific to
cyclin D1 or whether it was a function of cyclins in general, CV1 cells
were cotransfected with cyclin D1, E, or A at a 1:3 ratio of cyclin to
androgen receptor. As seen in Fig. 1C, PSA61LUC activation was not
significantly affected by cotransfection with cyclin E or A but was
inhibited by cyclin D1. All cyclin constructs have been previously shown
to encode functional cyclin gene products (11). Therefore, inhibition of
androgen receptor transactivation potential was not mediated by cyclin E
or cyclin A but was mediated by cyclin D1. To determine whether this
function of cyclin D1 was common to the D-type cyclin family, similar
experiments were performed using cyclin D3. As shown in Fig. 1D,
cyclin D3 was also capable of inhibiting PSA61LUC activation, albeit to
a lesser extent than cyclin D1. These results indicate that the ability to
inhibit androgen-mediated receptor transactivation potential was shared
by specific members of the D-type cyclin family.
Several classes of proteins have been reported to repress the transactivation function of the androgen receptor. Among these are dominant negative androgen receptor proteins, which lack transactivation
potential and prevent transactivation from wild-type receptors by
heterodimerization (14). We compared the transcriptional antagonistic
activities of two such mutant proteins, ARD38 –296 and ARD46 – 408,
with that exhibited by cyclin D1. At equivalent ratios of androgen
receptor:effector, all three inhibitory proteins reduced PSA61LUC
activation to just above background levels (Fig. 2). Therefore, transcriptional inhibition associated with cyclin D1 was similar to that
observed using known dominant negative androgen receptor proteins.
Transcriptional Repression Is Independent of the Cell Cycle
Function of Cyclin D. D-type cyclins play a significant role in cell
cycle progression, and activation of CDK4 complexes is imperative in
normal cells for progression into S phase. To address whether the transcriptional inhibition function of cyclin D1 is related to its role in cell
cycle progression, several approaches were used. First, simple reporter
assays using the androgen receptor and cyclin D1 were performed in
SAOS-2 cells. It has been shown that the only cell cycle function of
CDK4/cyclin D complexes is to phosphorylate and inactivate RB (15).
Because SAOS-2 cells do not express functional RB (16), they have lost
the requirement for CDK4/cyclin D, and unlike normal cells, overexpression of CDK4 and/or cyclin D in this cell line has no effect on cell cycle
progression. Although these cells are refractory to the cell cycle effects of
cyclin D1, androgen receptor transactivation potential was still repressed
in these cells by a 3:1 ratio of cyclin D1 to androgen receptor (Fig. 3A).
These data indicate that this attribute of cyclin D1 was independent of its
role in cell cycle progression.
To test this hypothesis, mutants of cyclin D1 were used that had
either lost the ability to bind to CDK4 (mutant CycD-KE) or to RB
(mutant CycD-GH; Ref. 17). As can be seen in Fig. 3B, these mutants
of cyclin D1 were able to suppress PSA61LUC activation as effectively as wild-type cyclin D1, again suggesting that this function of
cyclin D1 was independent of its cell cycle role. As such, it would be
predicted that cyclin D1 may bind to proteins other than CDK4 to
carry out this function, and so CDK4 could potentially then compete
for this function of cyclin D1. To test this, we cotransfected CDK4
along with cyclin D1 and the androgen receptor (3:3:1 ratio, respectively)
and monitored reporter activity. Strikingly, cotransfection of CDK4 was
able to partially restore the PSA61LUC activation that was inhibited in
the presence of cyclin D1 alone (Fig. 3C). In addition, cotransfection of
dominant-negative, kinase-defective CDK4 also partially restored
PSA61LUC activation. Taken together, the data in Fig. 3 suggest that
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Fig. 1. Cyclin D1 attenuates androgen receptor transactivation potential in a dose-dependent manner. CV1 cells propagated in the absence of androgen were cotransfected with:
CMV-b-galactosidase (CMV-betagal; 1 mg), an androgen receptor expression construct (AR0 or SG5-AR; 1 mg), the PSA61LUC reporter construct (1 mg), and either a cyclin expression
construct (3 mg) or empty vector RcRSV. After transfection, the precipitate was removed, and cells were either left untreated (f) or were treated with 0.1 nM R1881 (3) for 36 h.
After 36 h, cells were harvested, and luciferase activities were measured and normalized against b-galactosidase activity. Experiments were performed at least in triplicate. Columns,
averages; bars, SD. Maximum androgen receptor activity (as exhibited by cells transfected only with CMV-betagal, AR, PSA61LUC, and RcRSV and treated with R1881) was set
at 100%. A, androgen receptor transactivation potential is attenuated by cyclin D1. Cells transfected at a 1:3 ratio with either pAR0 or pSG5AR and cyclin D1 demonstrate reduced
transactivation potential. B, cyclin D1 attenuates androgen receptor transactivation in a dose-dependent manner. Cells were transfected either in the absence of cyclin or at a 1:1–1:5
ratio of androgen receptor to cyclin. C, attenuation of androgen receptor transactivation is specific to cyclin D. Cells were transfected with either cyclin D1, cyclin A, or cyclin E at
the ratios shown. D, transcriptional inhibition is conserved with other D-type cyclins. Cells were transfected with either cyclin D1 and cyclin D3 at the ratios indicated.
cyclin D1 inhibited androgen-mediated receptor transactivation potential
in a cell cycle-independent manner. Moreover, the data in Fig. 3C suggest
that the cyclin D1 complexes required for transcriptional squelching may
be distinct from CDK4-containing complexes.
Cyclin D1 Interacts Directly with the Androgen Receptor. Because of these observations and because of the functional relationship
we observed between cyclin D1 and the androgen receptor, we questioned whether these two proteins may physically interact. To test this,
we cotransfected CV1 cells at equal ratios with androgen receptor and
cyclin D1 expression plasmids. Transfected cells were lysed, and
protein complexes were recovered using antisera generated against
either cyclin D1, the androgen receptor, or MDM2 (negative control).
Immunoprecipitated complexes were subjected to SDS-PAGE and
immunoblotted for either the androgen receptor or cyclin D1. As can
be seen in Fig. 4A, complexes immunoprecipitated using anti-cyclin
D1 antisera contained both cyclin D1 and the androgen receptor.
Conversely, complexes immunoprecipitated using anti-androgen receptor antisera contained both the androgen receptor and cyclin D1
proteins. Lysates immunoprecipitated using anti-MDM2 antisera
failed to recover either cyclin D1 or the androgen receptor. To further
verify the specificity of this interaction, CV1 cells were cotransfected
with expression constructs for cyclin A and the androgen receptor, and
similar immunoprecipitation experiments were performed. As can be
seen in Fig. 4B, immunoprecipitates recovered using anti-androgen
receptor antisera contained only the androgen receptor protein and not
cyclin A. Conversely, immunoprecipitates recovered using anti-androgen
receptor antisera contained only the cyclin A protein and not the androgen
receptor. Lysates immunoprecipitated using anti-cyclin E antisera also
failed to recover either cyclin A or the androgen receptor (Fig. 4B). Thus,
cyclin D1 forms a specific complex with the androgen receptor.
Discussion
The data presented here demonstrate a new function for cyclin D1
in attenuating the transactivation potential of the androgen receptor.
Cyclin D1 inhibited transactivation from an androgen receptor-
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strate high levels of D-type cyclin expression and associated kinase
activity (11). On the basis of the studies presented here, we
propose that a negative feedback loop may exist in androgendependent prostate cells, wherein ligand-dependent activation of
the androgen receptor results in stimulation of cyclin D expression.
This net increase in cyclin D would promote cell cycle progression
but may also act to attenuate the transcriptional activity of the
androgen receptor, thus modulating the rate of future cell cycle
Fig. 2. Cyclin D-dependent transcriptional inhibition is similar to that observed with
dominant negative androgen receptors. CV1 cells were transfected as described in the
legend to Fig. 1 with the plasmids/ratios indicated. Constructs pSG5-rARD46 – 408 and
pSG5-rARD38 –296 encode known dominant negative AR proteins.
specific reporter, PSA61LUC, in a dose-dependent manner (Fig. 1).
The intensity of androgen receptor transcriptional inhibition was
comparable to that observed with known dominant negative androgen
receptors (Fig. 2). The ability to inhibit PSA61LUC transactivation
was shared with cyclin D3, but not with cyclin E or cyclin A (Fig. 1).
Interestingly, the ability of cyclin D1 to limit androgen receptor
transactivation potential was shown to be independent of the cell cycle
role of cyclin D because transcriptional repression was observed in
cells that are refractory to the growth-promoting effects of cyclin D1
and with mutants of cyclin D1 that are defective for binding to CDK4
or RB (Fig. 3). Also, the effect of cyclin D1 on PSA61LUC transactivation could be partially competed by overexpression of CDK4 or
kinase-defective CDK4 (Fig. 3). Finally, we observed a specific,
direct interaction between cyclin D1 and the androgen receptor, suggesting that the effect of cyclin D1 on androgen-dependent transactivation is direct (Fig. 4).
Our finding that cyclin D1 inhibits the transactivation potential of
the androgen receptor directly contrasts with its ability to activate
another steroid hormone receptor, the estrogen receptor, in a ligandindependent manner (2, 3). The finding that factors/proteins that act as
a coactivators for one nuclear receptor act as corepressors for another
receptor is not unprecedented. For example, the coactivator SRC-1
enhances transcriptional activity of many steroid hormone receptors
(e.g., estrogen, progesterone, and glucocortocoid receptors) but represses transcriptional activity of the androgen receptor (18). Breast
epithelial cells are dependent on estrogen for growth and, consequently, antiestrogens (e.g., 4-hydroxytamoxifen) are relied upon to
treat breast carcinoma (19). Because amplification or overexpression
of cyclin D1 is observed in .50% of breast carcinomas, this may
provide the means for breast cancer cells to bypass the estrogen
requirement (5, 20). Consistent with this and unlike estrogen-mediated activation, cyclin D1-induced activation of the estrogen receptor
is only slightly perturbed by 4-hydroxytamoxifen (2, 3). These studies
underscore the importance of understanding how hormone-dependent
cells regulate the requirement for steroids and how cancer cells evade
this requirement.
The studies presented here point to an important role for cyclin
D1 in regulating the transactivation potential of the androgen
receptor. We have previously shown that, in LNCaP cells, which
are dependent on androgen for growth, a decrease in D-type cyclin
expression is observed upon androgen withdrawal (11). Conversely, such cells cultured in the presence of androgen demon-
Fig. 3. Transcriptional inhibition is independent of the cell cycle function of cyclin D.
A, inhibition of androgen receptor transactivation potential is observed in SAOS-2 cells,
which are resistant to the growth-promoting activity of cyclin D. SAOS-2 cells were
transfected as in Fig. 1. B, mutants of cyclin D1 that have lost specific cell cycle functions
of cyclin D1 retain the ability to repress androgen receptor transactivation potential. CV1
cells were transfected as in Fig. 1 using wild-type cyclin D1, cyclin D1-GH (defective in
RB binding), or cyclin D1-KE (defective in binding or activation of CDK4) at the ratios
shown. C, androgen receptor transactivation potential can be partially restored by cotransfection with CDK4. CV1 cells were cotransfected as in Fig. 1 with constructs expressing
the androgen receptor, cyclin D1, and either wild-type CDK4 or a kinase-defective CDK4,
CDK4-K35M, at the ratios given.
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mutations of the RB/cyclin D1/p16ink4a pathway observed in human
breast versus prostate carcinomas.
Acknowledgments
We thank Drs. Erik Knudsen and William Biggs for invaluable suggestions
and critical discussions.
References
Fig. 4. Cyclin D1 associates with the androgen receptor. A, CV1 cells were transfected
with pSG5-AR (8 mg) and RSV-Cyclin D1 (8 mg) expression plasmids. Thirty-six h
posttransfection, cells were harvested and lysed, and clarified extracts were split for
immunoprecipitation with either anti-androgen receptor, anti-cyclin D1, or anti-MDM2
(control) antibodies. Immunoprecipitates were recovered using protein A-Sepharose,
washed using NETN, boiled, and resolved with input samples using 10.5% SDS-PAGE.
Proteins were then transferred to Immobilon, at which time the membrane was split: the
top half was probed using anti-androgen receptor antibodies, and the lower half was
probed using anti-cyclin D1 antibodies. B, CV1 cells were transfected with AR (8 mg) and
cyclin A (8 mg) expression plasmids. Thirty-six h posttransfection, cells were harvested
and lysed, and clarified extracts were split for immunoprecipitation with either antiandrogen receptor, anti-cyclin A, or anti-cyclin E (control) antibodies. Immunoprecipitates were recovered using protein A-Sepharose, washed using NETN, boiled, and
resolved with input samples in duplicate gels using 10.5% SDS-PAGE. Proteins were then
transferred to Immobilon, at which time one membrane was probed using anti-AR
antibodies, and the other was probed using anti-cyclin A antibodies.
progression. This hypothesis may explain the biphasic response of
prostate cells to androgen (21), wherein androgen-dependent prostate cells undergo growth arrest either in the absence of androgen
(i.e., in the absence of cyclin D) or in the presence of excess
androgen (i.e., in the presence of excess cyclin D).
Deregulation of cell cycle control is a common component of cancer.
Specifically, the RB/cyclin D1/p16ink4a pathway is targeted for disruption in .60% of studied tumors (1, 22). Typically, the mutations that
target this pathway are tumor type specific (1, 5, 23). For example, in
small cell lung carcinoma, RB is commonly inactivated, whereas in
non-small cell lung carcinoma, p16ink4a is lost. The basis behind this
specificity is largely not understood. The finding that cyclin D1 synergizes with the estrogen receptor indicates that the frequent amplifications
of cyclin D1 in breast cancer confer a specific growth advantage both
through ubiquitous activation of CDK4 and through activation of the
estrogen receptor. Our finding that cyclin D1 subverts androgen receptor
signaling provides an explanation for why cyclin D1 amplifications are
not commonly observed in prostate carcinomas, whether they are androgen dependent or androgen independent (24). In contrast, inactivating
mutations of RB are frequently observed in prostate carcinomas (25).
Together, these findings may help explain the cell type specificities of the
1. Sherr, C. J. Cancer cell cycles. Science (Washington DC), 274: 1672–1677, 1996.
2. Neuman, E., Ladha, M. H., Lin, N., Upton, T. M., Miller, S. J., DiRenzo, J., Pestell,
R. G., Hinds, P. W., Dowdy, S. F., Brown, M., and Ewen, M. E. Cyclin D1
stimulation of estrogen receptor transcriptional activity independent of cdk4. Mol.
Cell. Biol., 17: 5338 –5347, 1997.
3. Zwijsen, R. M. L., Wientjens, E., Klompmaker, R., van der Sman, J., Bernards, R.,
and Michalides, R. J. A. M. CDK-independent activation of estrogen receptor by
cyclin D1. Cell, 88: 405– 415, 1997.
4. Inoue, K., and Sherr, C. J. Gene expression and cell cycle arrest mediated by
transcription factor DMP1 is antagonized by D-type cyclins through a cyclin-dependent kinase independent mechanism. Mol. Cell. Biol., 18: 1590 –1600, 1998.
5. Palmero, I., and Peters, G. Perturbation of cell cycle regulators in human cancer.
Cancer Surv., 27: 351–367, 1996.
6. Resnitzky, D., Gossen, M., Bujard, H., and Reed, S. I. Acceleration of the G1/S phase
transition by expression of cyclins D1 and E with an inducible system. Mol. Cell.
Biol., 14: 1669 –1679, 1994.
7. Montie, J. E., and Pienta, K. J. Review of the role of androgenic hormones in the
epidemiology of benign prostatic hyperplasia and prostate cancer. Urology, 43:
892– 899, 1994.
8. Isaacs, J. T. Antagonistic effect of androgen on prostatic cell death. Prostate, 5:
545–557, 1984.
9. Trapman, J., and Brinkmann, A. O. The androgen receptor in prostate cancer. Pathol.
Res. Pract., 192: 752–760, 1996.
10. Pettaway, C. A. Prognostic markers in clinically localized prostate cancer. Tech.
Urol., 4: 35– 42, 1998.
11. Knudsen, K. E., Arden, K. C., and Cavenee, W. K. Multiple G1 regulatory elements
control the androgen-dependent proliferation of prostatic carcinoma cells. J. Biol.
Chem., 273: 20213–20222, 1998.
12. Chen, C., and Okayama, H. High efficiency transformation of mammalian cells by
plasmid DNA. Mol. Cell. Biol., 7: 2745–2752, 1987.
13. Cleutjens, K. B. J. M., van der Korput, H. A. G. M., van Eekelen, C. C. E. M., van
Rooij, H. C. J., Faber, P. W., and Trapman, J. An androgen response element in a far
upstream enhancer region is essential for high, androgen-regulated activity of the
prostate specific antigen promoter. Mol. Endocrinol., 11: 148 –161, 1997.
14. Palvimo, J. J., Ksllio, P. J., Ikonen, T., Mehto, M., and Janne, O. A. Dominant negative
regulation of trans-activation by the rat androgen receptor: roles of the N-terminal domain
and heterodimer formation. Mol. Endocrinol., 93: 1399 –1407, 1993.
15. Lukas, J., Parry, D., Aagaard, L., Mann, D. J., Bartkova, J., Strauss, M., Peters, G.,
and Bartek, J. Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour
suppressor p16. Nature (Lond.), 375: 503–506, 1995.
16. Templeton, D. J., Park, S. H., Lanier, L., and Weinberg, R. A. Nonfunctional mutants
of the retinoblastoma protein are characterized by defects in phosphorylation, viral
oncoprotein association, and nuclear tethering. Proc. Natl. Acad. Sci. USA, 88:
3033–3037, 1991.
17. Dowdy, S. F., Hinds, P. W., Louie, K., Reed, S. I., Arnold, A., and Weinberg, R. A.
Physical interaction of the retinoblastoma protein with human D cyclins. Cell, 73:
499 –511, 1993.
18. Ikonen, T., Palvimo, J. J., and Janne, O. A. Interaction between the amino- and
carboxyl-terminal regions of the rat androgen receptor modulates transcriptional
activity and is influenced by nuclear receptor co-activators. J. Biol. Chem., 272:
29821–29828, 1997.
19. Kimmick, G. G., and Muss, H. B. Endocrine therapy in metastatic breast cancer.
Cancer Treat. Res., 94: 231–254, 1998.
20. Bartkova, J., Lukas, J., Meuller, H., Leutzhft, D., Strauss, M., and Bartek, J. Cyclin
D1 protein expression and function in human breast cancer. Int. J. Cancer, 57:
353–361, 1994.
21. Sonnenschein, C., Olea, N., Pasanen, M. E., and Soto, A. M. Negative controls of cell
proliferation: human prostate cancer cells and androgens. Cancer Res., 49: 3474 –
3481, 1989.
22. Sellers, W. R., and Kaelin, W. G., Jr. Role of the retinoblastoma protein in the
pathogenesis of human cancer. J. Clin. Oncol., 15: 3301–3312, 1997.
23. Bartkova, J., Lukas, J., and Bartek, J. Aberrations of the G1- and G1/S-regulating
genes in human cancer. Prog. Cell Cycle Res., 3: 211–220, 1997.
24. Gumbiner, L. M., Gumerlock, P. H., Mack, P. C., Chi, S-G., deVere White, R. W.,
Mohler, J. L., Pretlow, T. G., and Tricoli, J. V. Overexpression of cyclin D1 is rare
in human prostate carcinoma. Prostate, 38: 40 – 45, 1999.
25. Phillips, S. M., Barton, C. M., Lee, S. J., Morton, D. G., Wallace, D. M., Lemoine,
N. R., and Neoptolemos, J. P. Loss of the retinoblastoma susceptibility gene RB1 is
a frequent and early event in prostatic tumorigenesis. Br. J. Cancer, 70: 1252–1257,
1994.
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D-Type Cyclins Complex with the Androgen Receptor and
Inhibit Its Transcriptional Transactivation Ability
Karen E. Knudsen, Webster K. Cavenee and Karen C. Arden
Cancer Res 1999;59:2297-2301.
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