Cyclin D1b variant influences prostate cancer growth
through aberrant androgen receptor regulation
Craig J. Burd*†, Christin E. Petre*†, Lisa M. Morey*, Ying Wang*, Monica P. Revelo‡, Christopher A. Haiman§, Shan Lu‡,
Cecilia M. Fenoglio-Preiser‡, Jiwen Li¶, Erik S. Knudsen*?, Jiemin Wong¶, and Karen E. Knudsen*?**
Departments of *Cell Biology and‡Pathology and Laboratory Medicine and?Center for Environmental Genetics, University of Cincinnati,
Cincinnati, OH 45267;¶Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030; and§Department of
Preventive Medicine, University of Southern California, Los Angeles, CA 90089
Edited by Michael G. Rosenfeld, University of California at San Diego, La Jolla, CA, and approved December 20, 2005 (received for review July 25, 2005)
Cyclin D1 is a multifaceted regulator of both transcription and
cell-cycle progression that exists in two distinct isoforms, cyclin
D1a and D1b. In the prostate, cyclin D1a acts through discrete
and thus limit androgen-dependent proliferation. Accordingly,
cyclin D1a is rarely overexpressed in prostatic adenocarcinoma and
holds little prognostic value in this tumor type. However, a com-
mon polymorphism (A870) known to facilitate production of cyclin
D1b is associated with increased prostate cancer risk. Here we
show that cyclin D1b is expressed at high frequency in prostate
cancer and is up-regulated in neoplastic disease. Furthermore, our
data demonstrate that, although cyclin D1b retains AR association,
it is selectively compromised for AR regulation. The altered ability
and in vivo assays and was associated with compromised regula-
tion of AR-dependent proliferation. Consistent with previous re-
ports, expression of cyclin D1a inhibited cell-cycle progression in
AR-dependent prostate cancer cells. Strikingly, cyclin D1b signifi-
cantly stimulated proliferation in this cell type. AR-negative pros-
tate cancer cells were nonresponsive to cyclin D1 (a or b) expres-
sion, indicating that defects in AR corepressor function yield a
growth advantage specifically in AR-dependent cells. In summary,
these studies indicate that the altered AR regulatory capacity of
cyclin D1b contributes to its association with increased prostate
cancer risk and provide evidence of cyclin D1b-mediated transcrip-
corepressor ? G870A ? polymorphism ? cell cycle ? thyroid hormone
PCa is based on their requirement for androgen (1). Current
therapies aim to reduce endogenous androgen production and?or
inhibit the activity of the androgen receptor (AR) (2) and are
by which AR activity is controlled is pivotal for improving PCa
AR is a member of the nuclear receptor superfamily and
(4). AR activation is initiated by ligand (androgen) binding, which
active receptor conformation (5). AR activation triggers nuclear
translocation, wherein AR binds specific DNA sequences termed
is enhanced by recruitment of coactivators that promote receptor
stabilization, facilitate DNA binding, or render DNA more acces-
sible to the transcriptional machinery (8). This action is opposed by
a select group of corepressor proteins that foster a state of inactive
Previously, we and others identified cyclin D1a as a critical AR
corepressor (9–11). Cyclin D1a is well characterized as a cell-cycle
rostate cancer (PCa) is the most frequently diagnosed malig-
nancy among men in the U.S., and treatment of disseminated
regulator, interacting with cyclin-dependent kinase (CDK)4?6 to
form an active kinase complex that promotes S-phase entry (12).
Accordingly, cyclin D1a is overexpressed in many tumor types, and
aberrant cyclin D1a–CDK4 activity leads to inappropriate prolif-
erative signaling (13). However, cyclin D1a is rarely overexpressed
in PCa and has no independent prognostic value in this tumor type
(14, 15). Mouse models of PCa demonstrate that cyclin D1a
expression is reduced as a function of tumor progression (16), and
cyclin D1a has been reported to be sequestered in the cytoplasm in
human disease (16). Together, these data suggest that cyclin D1a
function may negatively impact PCa growth and progression.
Emerging evidence suggests that this role of cyclin D1 is likely
attributed to cell-cycle-independent functions.
Cyclin D1a expression levels increase in response to androgen
stimulation in PCa cells, and subsequent CDK4 activation is essen-
tial for ensuing cell-cycle progression (17). However, accumulated
cyclin D1a binds directly to and inhibits AR, thus limiting further
cellular proliferation (9–11). This function of cyclin D1a is inde-
for cyclin D1a in transcriptional regulation (17–19). Cyclin D1a
association and regulation with a growing number of transcription
factors (e.g., thyroid hormone receptor ?, estrogen receptor ?,
signal transducer and activator of transcription 3, and Sp1) is
suspected to contribute to its function in tumorigenesis (18).
derived from PCa and liver, and this interaction significantly
represses ligand-dependent AR activity at equimolar ratios of AR
to cyclin D1a (11, 20, 21). Cyclin D1a transcriptional regulation of
the AR is manifested through at least two discrete mechanisms.
First, we and others have shown that cyclin D1a binds histone
deacetylase 3 (HDAC3) to mediate transcriptional repression (22,
23). Second, cyclin D1a directly binds the AR N terminus and
two cyclin D1a activities are effective in repressing AR transcrip-
tional potential on a wide spectrum of target genes (20). Recently,
the cyclin D1a repressor domain (RD) was mapped to a region
(amino acids 142–253) that is both required and sufficient for AR
inhibition and repression of androgen-dependent proliferation in
PCa cells (22). Combined, these data suggest that androgen-
induced elevation of cyclin D1a serves initially to stimulate cellular
proliferation; but upon CDK saturation, cyclin D1a uses discrete
mechanisms to attenuate subsequent AR activity. Supporting this
negative feedback hypothesis, AR activity is lowest at the G1?S
transition of the cell cycle, where cyclin D1a levels peak (24).
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: AR, androgen receptor; PCa, prostate cancer; ARE, androgen responsive
element; SLP, sex-limited protein; PSA, prostate-specific antigen; HDAC3, histone deacety-
lase 3; CDK, cyclin-dependent kinase; RD, repressor domain; PIN, prostatic intraepithelial
†C.J.B. and C.E.P. contributed equally to this work.
**To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
February 14, 2006 ?
vol. 103 ?
Despite the importance of cyclin D1a in the regulation of AR
been identified in PCa.
A polymorphism (G?A870) of the cyclin D1 locus has recently
been associated with increased cancer risk for a wide variety of
tumors, including PCa (25–27). Individuals harboring the A870
allele demonstrate an increased propensity to produce an alterna-
tively spliced cyclin D1 mRNA (transcript b). Translation of
transcript b results in the production of a variant of the cyclin D1a
protein, termed cyclin D1b, with a divergent C terminus (25).
Although individuals harboring the ‘‘A?A’’ genotype have an
increased propensity to produce transcript b, the levels of expres-
sion can be markedly variant and transcript a can still be detected
(25, 28). Given the discordance between genotype and cyclin D1b
production, assessment of genotype alone may not provide an
accurate assessment of risk. Recent investigations have demon-
strated that cyclin D1b harbors distinct functions from cyclin D1a
with regard to cell-cycle control and cellular transformation (29,
30). However, the influence of cyclin D1b on transcriptional
regulation had yet to be assessed.
Given the importance of cyclin D1 in modulating AR function,
we analyzed the prevalence and consequence of cyclin D1b in PCa.
We observed a striking representation of the A allele and cyclin
D1B production in both PCa cells and tumors. To delineate the
of this variant to regulate AR function was investigated. Using
multiple techniques, we demonstrate that although cyclin D1b
retains AR binding function it is selectively compromised for AR
regulation. The biological consequence of this defect is that cyclin
D1b stimulates androgen-dependent proliferation, in contrast to
the inhibition of cell-cycle progression mediated by cyclin D1a.
AR-negative PCa cells were refractory to regulation by both cyclin
D1 isoforms, demonstrating specificity of cyclin D1 action. To-
gether, these data suggest that cyclin D1b contributes to PCa risk
through compromised AR regulation and indicate that cyclin D1b
expression may yield a significant growth advantage for AR-
from cyclin D1a.
G?A870 Polymorphism and Cyclin D1b Expression. The A870 poly-
morphism of cyclin D1 is associated with increased PCa risk (26,
27). However, the prevalence of this genotype in established PCa
models and association with cyclin D1b production had yet to be
assessed. Initially, genotyping was performed by means of RFLP
from PCa cells (LNCaP, PC3, DU145, LAPC4, and 22RV1) and
two different PCa lymph node metastases (Met-1 and Met-2). For
a negative control, COS-7 cells were used, because this cyclin D1
locus does not amplify with human-specific primers. As shown in
Fig. 1A, Met-1 and three of the PCa cell lines (DU145, 22RV1, and
LAPC4) were homozygous for the A allele (Fig. 1A, lanes 6–9). A
heterozygous genotype was observed in Met-2, LNCaP, and PC3
cells (Fig. 1A, lanes 4, 5, and 10). These data demonstrate that the
A allele is predominant in PCa cell lines and tumor samples.
However, the A870 polymorphism does not necessitate transcript b
production. Specifically, it has been documented that individuals
harboring the AA genotype still produce cyclin D1a (25) and that
the G allele can generate cyclin D1b (25). Thus, determination of
transcript b expression is essential, because this parameter has
significantly increased the predictive value as compared with anal-
ysis of the genotype alone for some tumor types (28). To date, no
study has assessed the relative expression of cyclin D1b in PCa. To
monitor cyclin D1b expression, RT-PCR analysis of PCa cell lines
and tumors was performed by using primers specific for transcript
a or b (strategy shown in Fig. 7B). PCR amplification of pRC-
cyclin-D1b plasmid is shown as a positive control (Fig. 1B, lanes 3
transcriptase (Fig. 1B, lane 2). The four AA homozygous PCa
samples (DU145, LAPC4, 22RV1, and Met-1) exhibited increased
expression of cyclin D1b transcript (Fig. 1B, lanes 7–10) in relation
to their heterozygous counterparts (LNCaP, PC3, and Met-2) (Fig.
1B, lanes 5, 6, and 11). However, expression of both cyclin D1a and
D1 genotype is not predictive of the relative transcript ratio.
GAPDH was used as an internal control for all PCRs (Fig. 1B
Bottom). Further investigation into cyclin D1b expression was
performed by using tissue obtained from four different radical
prostatectomies and matching tissue from the same patient [clas-
sified to be normal or prostatic intraepithelial neoplasia (PIN), as
shown]. In addition, a third-lymph-node metastisis (Met-3) and an
immortalized prostatic epithelial cell line, HUNC-E, were also
analyzed for cyclin D1b expression. All tumor samples showed
cyclin D1b expression, consistent with previous results (Fig. 1C).
Interestingly, matched normal tissue and HUNC-E cells showed
lower expression of cyclin D1b, but PIN (precancerous lesions)
demonstrated increased expression. Taken together, these findings
in PCa models and confirm that the AA genotype does not predict
exclusive production of cyclin D1b.
Cyclin D1b Maintains Efficient AR Interaction. Given the importance
of cyclin D1a in the regulation of androgen-dependent prolifera-
attributes. As expected, cyclin D1b exhibited nuclear localization
and did not influence ligand-induced AR nuclear translocation
web site). Analysis of two AR-positive PCa lines with divergent
genotypes (A?G:LNCaP, and AA:22Rv1) confirmed endogenous
reverse transcriptase, was included as a control (lane 2). For each PCR, gener-
ated cDNA or cyclin D1b-encoding plasmid (lanes 3 and 4) was used in
GAPDH expression was performed as in B.
The AA genotype and transcript b are prevalent in PCa cells. (A) DNA
Burd et al.PNAS ?
February 14, 2006 ?
vol. 103 ?
no. 7 ?
cyclin D1b expression (Fig. 2A). Coimmunoprecipitation experi-
ments revealed an association between endogenous AR and cyclin
was recapitulated by using both ectopically expressed proteins (Fig.
8B) and in vitro binding assays (Fig. 8C). These findings revealed
that cyclin D1 exon 5 is not required for cyclin D1b binding,
consistent with maintenance of the RD (22) in cyclin D1b, and
identified cyclin D1b as a putative AR regulatory protein.
D1b associated directly with the AR (Figs. 2B, 8B, and 8C),
mechanistic similarity to cyclin D1a was investigated by assessing
the ability to recruit histone deacetylases and abrogate AR N?C-
terminal interaction (10, 21). The ability of cyclin D1b to regulate
AR N?C-terminal interactions was assessed by using a well char-
acterized mammalian two-hybrid system (Fig. 3A) (31). As previ-
ously described, cells were transfected with GAL4-AR628–919,
VP-16-AR1–565, Gal4-luciferase reporter, ?-gal, and expression
plasmid encoding cyclin D1a, D1b, cyclin D1?142–210, or vector
(21). There was no significant difference between the ability of
cyclin D1a and D1b to block the AR N?C interaction (Fig. 3A),
indicating that cyclin D1b harbors some AR regulatory ability. A
subdeletion (?142–210) of the RD that does not regulate N?C
interactions has no effect on this system. To ensure that cyclin D1
had no independent effect on the VP16 moiety, experiments were
repeated with a constitutively active Gal4-VP16 fusion. As shown
web site, ligand treatment and expression of cyclin D1a or D1b had
no effect on VP16 activity, demonstrating specificity of cyclin D1
action. To assess cyclin D1b association with HDAC3, cells were
transfected as indicated and lysates were immunoprecipitated with
antibodies specific for HDAC3, cyclin D1b, or control serum. A
specific interaction was observed between cyclin D1b and HDAC3
(Fig. 3B, lanes 2 and 4), and in vitro studies confirmed these results
(Fig. 8D). Together, these data verify that cyclin D1b functions to
recruit HDAC3 and inhibit AR N?C interactions. Thus, both
transcriptional regulatory functions of cyclin D1a are preserved in
the cyclin D1b variant.
Cyclin D1b Exhibits Compromised AR Corepressor Activity on the
Prostate-Specific Antigen (PSA) Promoter. Mounting evidence sug-
gests that the CDK-independent functions of cyclin D1a in tran-
scriptional regulation play important roles in cancer cells (18, 19).
However, the ability of cyclin D1b to exert these functions had yet
to be determined. Our data demonstrate that cyclin D1b harbors
activities that modulate transcription. To examine the functional
consequence of these activities in PCa cells, the impact on AR
activity was assessed. Initially, reporter assays were performed by
using the well characterized PSA-61-LUC reporter, which harbors
both an androgen-responsive proximal promoter and a distal
enhancer (32). As described, AR-negative cells were cotransfected
with a PSA reporter plasmid, AR, and increasing concentrations of
either HA-D1a or HA-D1b expression plasmid. Cyclin D1a was a
potent, dose-dependent inhibitor of AR action (Fig. 3C) (21),
consistent with previous reports. Specificity of this action was
demonstrated in that a cyclin D1 mutant incapable of binding AR
(cyclin D1-?RD) had no impact on PSA promoter activity (ref. 22
and data not shown). However, cyclin D1b was significantly com-
promised for repression of this AR target. As shown, AR activity
was elevated between 2- and 5-fold in cyclin D1b as compared with
cyclin D1a-expressing cells (Fig. 3C). The difference in AR atten-
uation was not due to decreased expression of cyclin D1b (Fig. 3C
Inset). These results show that cyclin D1b exhibits compromised
ability to regulate AR function at the PSA locus. Further investi-
gation into the effects of cyclin D1a and D1b expression on the
endogenous PSA locus was performed by using quantitative real-
whereas cyclin D1a expectedly reduced PSA mRNA levels. By
contrast, cyclin D1b was significantly compromised for regulating
PSA expression, thus validating the studies performed on ectopic
Cyclin D1b Regulates Transcription in a Manner Distinct from Cyclin
D1a. Because cyclin D1b demonstrated weakened repressor capa-
bility on the PSA-61-LUC reporter, the specificity of this effect was
cyclin D1b in transfected C33A cells or endogenous expression in LNCaP and
22Rv1 cells. (B) Lysates from LNCaP and 22RV1 cells were immunoprecipitated
with antibodies specific for AR, cyclin D1b, or a nonspecific control. The
precipitated complex was immunoblotted for AR expression.
Cyclin D1b associates with the AR in PCa cells. (A) Immunoblot of
AR N?C-terminal interactions was assessed in CV1 cells transfected with Gal4-
(Top), and cyclin D1a, cyclin D1b, or vector. After transfection, cells were
treated for 24 h with either vehicle or 5th-dihydrotestosterone (DHT) as
Results shown represent the average induction with standard deviations (?,
as in Fig. 2. (C) CV1 cells were transfected as indicated and analyzed in A.
Relative DNA used is shown. (Inset) Relative expression of AR- and HA-tagged
proteins transfected at a 1:3 ratio. (D) LNCaP cells were transfected with
pBABE-Puro and pCDNA, cyclin D1a, cyclin D1b, or cyclin D1-?RD. mRNA from
puromycin resistant cells was harvested and subjected to quantitative real-
time RT-PCR. Data represent the average of at least three independent points
performed in triplicate with standard deviations.
Cyclin D1b is compromised for AR corepressor activity. (A) Impact on
www.pnas.org?cgi?doi?10.1073?pnas.0506281103Burd et al.
assessed. Experimental conditions were first validated by using the
wherein cyclin D1b was inefficient as a repressor (similar to Fig. 3
C and D). Identical conditions were used to assess cyclin D1b
impact on the PSA-LUC reporter, which lacks the distal enhancer
region and associated AREs (33). Again, cyclin D1b failed to
repress AR activity to the capacity of cyclin D1a (Fig. 4A). To
analyze a larger spectrum of validated AR targets, cyclin D1b-
mediated regulation of probasin (ARR2) and sex-limited protein
(SLP) expression was assessed (33, 34). As shown, SLP-HRE2-
PSA promoter, resulting in a 2.2-fold increase in AR activity as
compared with cyclin D1a (Fig. 4A). Thus, cyclin D1b was com-
promised for AR regulation on multiple targets. By contrast, cyclin
D1b showed a statistically significant increase (?16%) in repressor
activity on the probasin (ARR2-LUC) reporter when compared
with cyclin D1a (Fig. 4A). These data indicate that cyclin D1b
harbors altered transcriptional activities as compared with cyclin
D1a and also demonstrate that the poor repressor functions ob-
served with PSA and SLP regulation cannot be attributed to
nonspecific effects. Last, no difference in AR corepression was
observed on the MMTV-LUC reporter, which contains three
nuclear receptors (e.g., GR) (Fig. 4B) (35). For MMTV regulation,
the data clearly show that cyclin D1a and cyclin D1b activities are
indistinguishable. The cyclin D1-?RD construct had no effect on
SLP or MMTV activity, as expected and consistent with our
previous observations (ref. 22 and data not shown). Interestingly,
cyclin D1b demonstrated an increased ability to repress thyroid
hormone receptor ? (Fig. 10, which is published as supporting
information on the PNAS web site). Together, these data demon-
strate that cyclin D1b can regulate transcription and that its ability
to modulate AR activity is altered as compared with cyclin D1a.
To validate this hypothesis, a Xenopus oocyte system was used.
37). In this system, reporter DNA introduced into the nuclei of
coupled (ssDNA template) or replication-independent (dsDNA)
pathway in a template-dependent manner (38). We exploited this
system to examine the relative effect of cyclin D1a and D1b on AR
activation as depicted (Fig. 5A). For protein expression, the indi-
cated mRNAs and subsequent reporter constructs (MMTV-LTR-
CAT or PSA-61-LUC) were injected into the oocyte cytoplasm.
expression and effect of cyclin D1a and D1b on AR-dependent
transcription was revealed by immunoblotting (Fig. 5B) and primer
extension (Fig. 5 C and D), respectively. As shown, AR activity on
the MMTV-CAT reporter was enhanced ?14-fold with the addi-
tion of R1881 and inhibited by the addition of either cyclin (Fig.
5C). However, cyclin D1a was 35% more effective as a repressor
than cyclin D1b on this promoter. Similar results were observed on
PSA-61-LUC, wherein cyclin D1a was 63% more effective than
D1b (Fig. 5D). Thus, cyclin D1b repressor activity was compro-
mised on both AR targets in vitro but again demonstrated a more
severe defect on the PSA-61-LUC. Combined, these data confirm
that cyclin D1b regulates transcription in a manner distinct from
Cyclin D1b Fails to Repress Androgen-Dependent Growth. Our data
indicate that, although cyclin D1b retains some AR modulatory
function, its corepressor activity is significantly altered. We have
previously shown that elevated cyclin D1a inhibits endogenous AR
activity and AR-dependent proliferation (10, 22). Therefore, we
studies, AR-positive?AR-dependent LNCaP cells were transfected
with the indicated expression plasmids and allowed to recover
with previous studies, cyclin D1a expression inhibited proliferation
(14.2% as compared with control) in androgen-dependent PCa
cyclin D1b failed to repress proliferation and actually stimulated
cell-cycle progression in these cells (8.2%) (Fig. 6A). Growth
regulation by both cyclin D1a and D1b was also monitored in the
AR-negative PCa cells (PC3), wherein no significant difference in
proliferation was noted upon ectopic expression of either cyclin
(Fig. 6B). These data demonstrate the specificity of cyclin D1b
on androgen-dependent PCa growth. Combined, these data indi-
Reporter assays were performed as in Fig. 3C by using ARE-containing pro-
moters (*, P ? 0.05;***, P ? 0.001). (B) CV1 cells were transfected as in A by
using the MMTV-LUC reporter (?, P ? 0.05).
Cyclin D1b displays promoter-specific regulation of the AR. (A)
in vitro. (A) Xenopus oocytes were injected and treated as shown here and as
for AR or cyclin expression by anti-flag immunoblot. (C and D) Oocytes were
injected and treated as depicted in A with either MMTV-CAT or PSA-61-LUC.
Cyclin D1b demonstrates promoter-specific repression of AR activity
Burd et al. PNAS ?
February 14, 2006 ?
vol. 103 ?
no. 7 ?
cate that cyclin D1b expression may render a specific growth
advantage in AR-positive PCa cells.
that the A allele is present at high frequency in all tested PCa cell
lines and tumor tissues and that transcript b was expressed in all
tumor and PIN tissues tested (Fig. 1). Moreover, although cyclin
D1b was found in association with AR in PCa cells (Fig. 2), AR
modulation was markedly distinct from that observed with cyclin
D1a. First, cyclin D1b was selectively compromised for regulation
of multiple AR target genes (Figs. 3–5). Second, the biological
consequence of this disparity was revealed in that, whereas cyclin
D1a significantly attenuated androgen-dependent proliferation in
effect on proliferation was observed in AR-negative PCa cells (Fig.
6B), indicating that regulation of androgen-dependent growth is
mediated through the AR. Combined, these data are the first to
identify a transcriptional regulatory function of cyclin D1b and
demonstrate that cyclin D1b harbors activities distinct from cyclin
D1a. Moreover, these studies suggest that cyclin D1b expression
may yield a significant growth advantage in AR-positive PCa cells,
thus revealing one potential mechanism by which the A870 allele
may alter PCa risk.
Cyclin D1b Selectively Modulates AR Activity. A major finding of this
prostate and PCa cells and that cyclin D1b selectively regulates AR
function. Numerous AR comodulators have previously been re-
ported to demonstrate promoter specificity, although the underly-
ing mechanism behind such action is poorly understood. For
on promoters containing minimal AREs, yet they fail to repress
probasin expression (39). In addition, N?C-terminal AR interac-
tions are required for activity of the full-length PSA and probasin
in AR regulation by cyclin D1a versus D1b was observed both in
mammalian cell assays and on chromatinized reporters in Xenopus
oocytes, wherein the ratio of cyclin D1 repression (D1a?D1b) on
the PSA-61-LUC promoter was twice as high as on the MMTV
promoter (Fig. 5). ARE sequence may play a role in determining
promoter specificity, because AR binding affinity is response-
element-specific (41). As shown, cyclin D1a effectively represses
(Figs. 3–5) (20). In contrast, cyclin D1b displayed compromised
ability to regulate the AR. It is unlikely that N?C-terminal inter-
actions play a role in cyclin D1b promoter specificity, because the
AR activation of both the SLP and MMTV promoters does not
require this association (40). Because the AR inhibitory activity of
cyclin D1a and site of HDAC3 recruitment have been previously
mapped to RD (22), and cyclin D1b maintains this region, it is not
the relative recruitment and requirement of HDAC3 at each target
promoter has yet to be addressed. Future studies will be needed to
target gene expression.
Cyclin D1b Demonstrates Altered Regulation of Androgen-Dependent
Proliferation. Previous studies report that cyclin D1a repression of
AR activity modulates androgen-dependent PCa proliferation. As
such, the presence of a negative feedback loop has been proposed
rate of further cell-cycle progression (9, 10, 17, 22). This hypothesis
that AR activity is inversely correlated with cyclin D1 expression
(24), that cyclin D1 expression is lost as a function of prostate
PCa progression in the transgenic adenocarcinoma of the mouse
be sequestered to the cytoplasm in advanced prostate tumors (16).
proliferation in AR-positive PCa cells, but actually stimulates
cell-cycle progression in this cell type. The data presented suggest
that the altered transcriptional regulatory functions of cyclin D1b
influence its affect on proliferation and that cyclin D1b expression
studies revealed that cyclin D1b was ineffective at repressing AR
activity on selected AR targets, including one used clinically to
monitor PCa progression (PSA). By contrast, cyclin D1b demon-
strated enhanced corepressor activity on a target involved in a
developmental process (SLP) and on a thyroid hormone receptor-
specific target. Together, these data indicate that the specificity of
cyclin D1b action may act disparately on specific classes of AR
target genes. Unfortunately, relatively few direct AR target genes
have been identified and validated, and the principle targets
involved in mediating androgen-dependent proliferation remain
elusive. Thus, the present study indicates that it will be essential to
identify the specific cohort of AR target genes that are disparately
regulated by both cyclin D1a and cyclin D1b.
In summary, our study highlights the importance of analyzing
cyclin D1b expression and function in PCa. We show that the
A870G polymorphism is present and that transcript b is produced
as a modifier of gene transcription and demonstrate that this
function of cyclin D1b may have critical consequence for cancer
cells. We show that cyclin D1b is selectively compromised for AR
growth advantage to AR-positive PCa cells. This study reveals a
facet of cyclin D1b function and puts forth a potential mechanism
through which cyclin D1b may alter PCa risk. Given the ability of
types, these data also provide the impetus to determine the
contribution of transcriptional control in the analysis of cancer-
related cyclin D1b activity.
Materials and Methods
Cell Culture. PCa cells were obtained and cultured as described (10,
20, 22). LAPC4 cells were obtained from C. Sawyers (University of
California, Los Angeles) (42). For steroid-free conditions, phenol
red free media was used with charcoal dextran-treated serum.
Tumor Sample Collection. Three inguinal lymph nodes with meta-
static prostate carcinoma and four radical prostatectomy tissues
were obtained from University Hospital (Cincinnati). After patho-
logical examination, fresh samples were divided into 1-cm3pieces
and immediately frozen.
Plasmids. Most plasmid constructs used were previously described
(20–22, 30, 31, 33, 36). pCDNA-HA D1b was amplified by PCR
polymorphism. (A) LNCaP cells were transfected with H2B-GFP alone, H2B-GFP
each of at least six coverslips. Bars represent relative percent BrdUrd incorpora-
tion, and error bars represent the standard deviation. (B) PC3 cells were trans-
compared with vector; ?, P ? 0.05 compared with vector).
www.pnas.org?cgi?doi?10.1073?pnas.0506281103 Burd et al.
flanked by XbaI and XmaI restriction sites and inserted into the Download full-text
corresponding sites of MS2-Flag. pRC-D1a was amplified by PCR
flanked by BamHI sites. The resulting PCR product was inserted
into MS2-Flag. The integrity of all constructs was verified by
sequencing. SLP-HRE2-LUC was a generous gift from F. Claus-
sens (University of Leuven, Louvain, Belgium) and was described
in ref. 34.
PCR-RFLP Analysis. Genomic DNA was extracted from cell culture
lines and tumor tissues by using the DNAeasy purification kit
(Qiagen, Valencia, CA). Isolated DNA was quantified, and 300 ng
of DNA was used in a PCR as described (43). Products were
extensively digested with ScrFI and resolved by agarose gel elec-
RT-PCR. RNA was isolated by using TRIzolfromculturedcelllines
or isolated tumor tissue. PCR amplification of cyclin D1b and
GAPDH was performed as described (20, 43). Cyclin D1a ampli-
for 30 cycles (94°C for 1 min, 51°C for 1 min, 72°C for 1 min).
Resulting products were resolved by agarose gel electrophoresis.
Real-time PCR was performed on cDNA prepared from LNCaP
cells transfected with indicated plasmids and pBabe-puro by means
of the Lipofecten method. Transfected cells were rapidly selected
in puromycin followed by RNA extraction. Applied Biosystems
expression and quantitated via the ??Ct method as per manufac-
turer’s protocol using the Applied Biosystems 7500F PCR system.
Reporter Assays. CV1 cells were seeded in steroid-free conditions
and transfected as indicated with a total of 4 ?g of DNA by using
the calcium phosphate method (44). For each transfection, 0.5 ?g
of CMV-?-gal, 0.75 ?g of reporter, and 0.5 ?g of SG5-AR were
added. Cyclin D1a, cyclin D1b, or pCDNA (empty vector) con-
structs were included as indicated. After transfection, cells recov-
ered for 18 h before stimulation with either hormone (0.1 nM
itored for luciferase and ?-gal (internal control for transfection
efficiency) activity as described (10). Data shown represent the
average and standard deviation of a minimum of six independent
data points. Appropriate P values were obtained by using ANOVA
followed by Newman–Keuls multiple-comparison post tests.
Cell Proliferation Assays. For BrdUrd assays, cells were seeded on
coverslips and transfected with 2 ?g of GFP-D1a, GFP-D1b, cyclin
D1-?RD?H2B-GFP, or H2B-GFP alone by means of Lipofectin.
BrdUrd staining was performed as described (22).
Protein–Protein Interaction Studies. Immunoprecipitation assays
were performed as described by using antibodies specific for AR
(22), HDAC3 (H-99, Santa Cruz Biotechnology), cyclin D1a
(AB-3, NeoMarkers), cyclin D1b (described in Fig. 11, which is
published as supporting information on the PNAS web site), and
preimmune serum as the nonspecific control. Immunoblotting of
precipitated complexes was performed with identical antibodies,
with the exception of HDAC3 (B12, Santa Cruz Biotechnology). In
vitro binding assays were performed as described (10).
Transcriptional Analyses in Xenopus Oocytes. cDNAs for AR, cyclin
as described (36). mRNA was generated by using linearized DNA
templates and an SP6 Message Machine kit. The preparation of
stage VI Xenopus oocytes and microinjection were performed
essentially as described (36). For transcriptional analysis, individual
or mixture of mRNAs were injected into the cytoplasm of the
oocytes for protein synthesis (500 ng??l for AR and 250 ng??l
and 500 ng??l for D1a and D1b, 18.4 nl per oocyte), and followed
2–3 h later by nuclear injection of the MMTV-CAT or PSA-Luc
reporter (50 ng??l, 18.4 nl per oocyte). Injected oocytes were
incubated at 18°C overnight in modified Barth’s solution in the
presence or absence of 50 nM R1881. Transcriptional analysis by
primer extension was performed essentially as described (37) by
using a CAT-specific or luciferase-specific primer for detection of
transcripts from the MMTV-CAT or PSA-LUC reporter. As an
internal control, primer extension of the endogenous histone H4
mRNA was performed as described (37).
We thank the K.E.K. and E.S.K. laboratories for critical reading of the
manuscript. This work was supported by National Institutes of Health
(NIH) Grants CA 099996 and CA 093404 (to K.E.K.), the Center for
Environmental Genetics (NIH?National Institute on Environmental
Health Sciences Grant P-30-ES06096), NIH Grant CA106471 (to
E.S.K.), NIH Grant DK065264 (to J.W.), the Albert J. Ryan Foundation,
and NIH Training Grant T32 ES07250-16.
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