Prostate cancer progresses from a hormone-sensitive, androgen-dependent
stage to a hormone-refractory, androgen-independent tumor. The androgen receptor
pathway functions in these androgen-independent tumors despite anti-androgen
therapy. In our LAPC-4 prostate cancer model, androgen-independent sublines
expressed higher levels of the HER-2/neu receptor tyrosine kinase than their
androgen-dependent counterparts. Forced overexpression of HER-2/neu in androgen-dependent
prostate cancer cells allowed ligand-independent growth. HER-2/neu activated
the androgen receptor pathway in the absence of ligand and synergized with
low levels of androgen to 'superactivate' the pathway. By modulating the response
to low doses of androgen, a tyrosine kinase receptor can restore androgen
receptor function to prostate cancer cells, a finding directly related to
the clinical progression of prostate cancer.
Prostate cancer begins as an androgen-dependent tumor that undergoes clinical
regression in response to pharmacological or surgical strategies that reduce
testosterone concentration. Despite this treatment, the cancer eventually
regrows as an androgen- or hormone-independent tumor. The molecular basis
for hormone independent cancer progression is poorly understood. Most androgen-independent
prostate tumors continue to express androgen receptor (AR) as well as the
androgen-dependent gene prostate-specific antigen (PSA), which indicates that
these cells maintain a functional AR signaling pathway despite castrate levels
of testosterone. Recent attention has focused on the hypothesis that AR itself
mediates androgen-independent progression.
There are two variations of this hypothesis. One is that amplification
of AR or mutation in the hormone binding domain, which occur in 20−30%
of androgen-independent prostate tumors1,
2,
3, alter its function.
In at least one example, mutation alters the specificity of the ligand binding
domain such that the mutant AR can bind and respond to other steroid hormones
such as estrogen4. An alternative model is that recruitment
of non-steroid receptor signal transduction pathways activate AR in the setting
of clinical androgen deprivation. The progesterone and estrogen receptors
(ER) can be activated by epidermal growth factor5 (EGF), dopamine6, insulin-like growth factor 1 (IGF-1)(7)
and cAMP (8). Activation of ER occurs by
phosphorylation at Ser 118 through the mitogen-activated protein kinase pathway9. AR can also become activated in a ligand-independent manner by
IGF-1, EGF and keratinocyte growth factor10 (KGF), but the mechanistic
details are unknown. The implications of these observations for human disease
remain unclear.
HER-2/neu, a member of the EGF family of receptor tyrosine kinases, is
overexpressed in 20−30% of human breast and ovarian cancers11.
Several observations indicate interactions between HER-2/neu and ER signaling
in breast cancer. Overexpression of HER-2/neu in breast cancer is inversely
correlated with ER levels12,
13 and predicts clinical resistance
to the anti-estrogen tamoxifen14,
15. Forced expression of HER-2/neu
induces ER activation, tyrosine phosphorylation and confers estrogen-independent
growth16, indicating that there is cross-talk between HER-2/neu
and ER. HER-2/neu may also be involved in prostate cancer. HER-2/neu is normally
expressed in prostate epithelial cells17,
18, and the heregulin
ligand is expressed in the stroma and basal epithelial cells of the normal
prostate gland19. In some but not all studies, HER-2/neu is
overexpressed and/or amplified at the DNA level in a subset of prostate cancer
patients20,
21 and has been associated with shortened survival22,
23. It is difficult to evaluate the frequency of HER-2/neu abnormalities
in advanced prostate cancer, as these tissues are not routinely biopsied.
However, elevated serum levels of Her2 extracellular domain have been correlated
with hormone-refractory disease after endocrine therapy24.
We have established androgen-dependent human prostate cancer xenografts
and developed androgen-independent sublines25. During studies
of differential gene expression between androgen-dependent and androgen-independent
sublines, we noted a consistent increase in HER-2/neu protein levels in association
with progression to androgen-independence in the LAPC-4 line. Forced overexpression
of HER-2/neu in androgen-dependent prostate cancer cells was sufficient to
confer androgen-independent growth in vitro and accelerate progression
to androgen-independence in castrate animals. HER-2/neu activated the AR signaling
pathway in the absence of ligand and enhanced the magnitude of AR response
in the presence of low levels of androgen. Reconstitution experiments established
that the effects of HER-2/neu on the AR pathway require expression of AR.
These findings demonstrate that there is cross-talk between the HER-2/neu
and AR pathways, and provide mechanistic insight into the clinical problem
of androgen-independent prostate cancer progression.
Increased HER-2/neu in androgen-independent xenografts We determined whether HER-2/neu was differentially expressed in androgen-dependent
and androgen-independent sublines of prostate cancer xenografts. We have derived
several androgen-independent sublines from the original androgen-dependent
LAPC-4 xenograft25 by castrating male SCID mice with androgen-dependent
tumors, waiting for regrowth of androgen-independent tumors, then serially
passaging androgen-independent xenografts. The level of HER-2/neu protein
expression was increased from 2-fold to 25-fold, with a trend towards enhanced
HER-2/neu expression with serial passaging (Fig. 1a).
We found reduced but detectable levels of AR protein in all androgen-independent
LAPC-4 sublines that overexpress HER-2/neu (Fig. 1b),
analogous to the observation that breast cancers overexpressing HER-2/neu
have reduced levels of ER protein12,
13. Androgen independence
was not caused by mutations in AR, as no mutations were found in the sequences
of the ligand-binding domain of AR from androgen-independent LAPC4 tumors
(data not shown). Therefore, androgen-independent growth is associated with
increased levels of HER-2/neu and reduced levels of AR in the LAPC-4 model.
Figure 1. Expression of HER-2/neu in androgen-dependent and androgen-independent
sublines of human prostate cancer xenografts.
Immunoblot analysis of whole-cell lysates from matched androgen-dependent
(AD) and androgen-independent (AI) sublines of the LAPC-4 human prostate cancer
xenograft, for expression of the 185-kDa HER-2/neu protein (a; -Her2)
or the 110-kDa androgen receptor protein (b; -AR). This short
exposure demonstrates the difference in HER-2/neu expression between AD and
AI samples; longer exposure confirmed expression of low levels of HER-2/neu
protein in all androgen-dependent LAPC-4 sublines (not shown). Equal loading
and transfer of protein to the immunoblot filter was confirmed by Ponceau
S staining for total protein (not shown).
HER-2/neu causes androgen-independent growth If HER-2/neu affected the AR signaling pathway, then overexpression should
promote androgen-independent growth in vitro and in vivo. To
test this, we measured the effects of HER-2/neu overexpression in the androgen-dependent
prostate cancer cell line LNCaP. We found that the growth rate of LNCaP cells
in culture was reduced by more than 50% after 48 hours in androgen-depleted
serum, as expected26. Addition of dihydrotestosterone restored
growth to levels seen with complete media (Fig. 2b).
Whereas LNCaP cells transformed with the neo vector alone showed a 42% decrease
in growth in androgen-deprived medium, two independent subclones of LNCaP
cells infected with a retrovirus overexpressing HER-2/neu protein (LH2-K,
LH2-N) (Fig. 2a) showed only a 15% decrease (Fig. 2c). Thus, HER-2/neu can partially rescue LNCaP
cells from growth arrest induced by androgen deprivation in vitro.
Figure 2. Effect of HER-2/neu on growth of androgen-dependent prostate cancer
cells in vitro in the absence of androgen.
Androgen-dependent LNCaP prostate cancer cells were infected with retrovirus
expressing the control Neo gene or HER-2/neu, and stable lines (LN-Neo and
LH2-N, respectively) were derived by selection in G418. a, Immunoblot
analysis of HER-2/neu (p185Her2) and androgen receptor (AR) expression. Endogenous
levels of HER-2/neu protein expression were observed in LN-Neo cells with
longer exposure (not shown). b, The effect of androgen on growth of
parental LNCaP cells. Cells were plated in media supplemented with either
10% fetal bovine serum (FBS) or 10% charcoal-stripped FBS with 0 nM, 1 nM
or 50 nM dihydrotestosterone (DHT), and MTT assays were done after 48 h. Data
represent mean absorbance readings at 540 nM s.d. from triplicate
wells. c, LN-Neo and LH2-N cells were plated in media supplemented
with either 10% FBS () or 10% charcoal-stripped FBS (), and
MTT assays were done after 48 h. MTT values for charcoal-stripped FBS are
expressed as % relative to complete FBS (representative experiment from seven
independent experiments).
We measured the effects of HER-2/neu on the in vivo growth of LNCaP
cells in castrated male mice, in which the residual androgen level is insufficient
to maintain growth of androgen-dependent prostate cancer cells25,
27,
28.
Intact or castrated male SCID mice injected subcutaneously with LNCaP/Neo
or LNCaP/HER-2 cells were examined weekly for evidence of tumor formation
and scored as positive when tumors greater than 0.5 cm in diameter were detected.
HER-2/neu conferred a modest growth advantage in intact male mice (
Fig. 3a), but shortened the latency for tumor formation by 50%
(from 30 to 15 weeks) in castrated males (Fig. 3b).
Tumors expressing HER-2/neu were also larger and produced higher levels of
circulating PSA in the serum (data not shown). Thus, HER-2/neu can substitute
for androgen to cause prostate cancer cells to grow in vivo.
Figure 3. Effect of HER-2/neu overexpression on tumorigenicity of prostate cancer
cells in intact and castrated male SCID mice.
Parental LNCaP cells () or sublines that overexpress HER-2/neu (LH2-N, ;
and LH2-K, ) were injected into intact (a) and castrated (
b) male mice (n = 8 for each condition). Tumor size was measured
weekly by calipers, and tumors were scored as positive when greater than 0.5
cm in any dimension. Data are presented as % of animals that developed a tumor
versus time. Serum PSA levels were comparable (data not shown).
Increased PSA levels in HER-2/neu-expressing prostate cells The ability of HER-2/neu overexpression to cause androgen-independent growth
in prostate cells might be explained by activation of the AR signaling pathway
in a ligand-independent fashion. If the model is correct, there should be
evidence of AR activation in cells expressing HER-2/neu in the presence of
low concentrations or the complete absence of androgen. We assessed the effects
of HER-2/neu on the expression of PSA, a well-characterized prostate-specific
gene whose transcription is strictly regulated by androgen29.
Immunoblot analysis demonstrated an increase in endogenous PSA protein in
lysates from LNCaP cells overexpressing HER-2/neu (Fig. 4
a). LNCaP/HER-2 cells also secreted sixfold to sevenfold more PSA
than did LNCAP/Neo cells, and this level was enhanced by the addition of R1881
(Fig. 4b). These findings demonstrate that activation
of the AR-responsive PSA gene mediated by HER-2/neu does not require exogenously
added androgen, and that HER-2/neu augments PSA secretion in response to androgen,
indicating that there may be cross-talk between the HER-2/neu and AR pathways.
Figure 4. Effect of HER-2/neu on levels of the androgen-regulated PSA protein.
a, Immunoblot analysis of whole-cell lysates from LNCaP cells expressing
the control Neo gene (LN-Neo) or HER-2/neu (LH2-N), for expression of PSA
protein. This short exposure demonstrates the effect of HER-2/neu expression
on PSA protein levels; longer exposure confirmed expression of PSA protein
in LN-Neo cells (not shown). Equal loading and transfer of protein to the
immunoblot filter was confirmed by Ponceau S staining for total protein (not
shown). b, PSA protein concentration in supernatant was measured by
ELISA after 24 h of exposure to serum-free media with (+) or without (−)
1 nM R1881. Data are expressed as fold increase relative to LN-Neo cells in
the absence of R1881; the actual concentration of PSA protein in LN-Neo cells
without R1881 was 0.78 ng/ml. The human kidney epithelial cell line 293 served
as a negative control and failed to secrete any detectable PSA protein.
HER-2/neu enhances PSA transcription The PSA promoter/enhancer contains high-affinity AR binding sites and functions
in an androgen-dependent manner30,
31. To measure the effects
of HER-2/neu on this response, we co-transfected LNCaP cells with the PSA-P/E-luc
reporter32 and HER-2/neu or the empty vector, then cultured
the cells in phenol red-free media in the absence of serum to allow precise
control of androgen concentration. In four independent experiments, HER-2/neu
activated the PSA promoter/enhancer construct sixfold to sevenfold in the
absence of added androgen (Fig. 5a). LNCaP cells
contain a mutation in the AR hormone binding domain that can alter AR function.
To eliminate the possibility of any effect of this mutation on the HER-2/neu
response, we used the androgen-dependent prostate cancer cell line LAPC-4,
which contains no mutations in exons 2−8 of AR (25). HER-2/neu activated the PSA promoter/enhancer 15-fold
in LAPC-4 cells in the absence of androgen (Fig. 5a).
Next, we assessed the effect of HER-2/neu in combination with androgen. R1881
activated PSA-P/E-luc 3-fold to 50-fold in LNCaP/Neo cells at concentrations
of 0.03, 0.1 and 1.0 nM (Fig. 5b). In the absence
of R1881, HER-2/neu activated the PSA-P/E-luc reporter sevenfold, and this
response was enhanced at all doses of androgen tested (Fig.
5b), indicating that HER-2/neu activates the PSA promoter/enhancer
in the absence of androgen but does not prevent further responsiveness to
androgen.
Figure 5. Effects of HER-2/neu on androgen-dependent PSA transcription.
a, Androgen-dependent LNCaP or LAPC-4 cells transfected with the
PSA P/E-luc reporter plasmid (bottom) in conjunction with a Neo ()
or HER-2/neu () expression vector were plated in serum-free, phenol
red-free media. A plasmid expressing GFP was included as a transfection control.
Luciferase activity was measured after 48 h. Data (a representative experiment
from a total of four) are expressed as fold activation relative to the luciferase
activity in LNCaP or LAPC-4 cells transfected with Neo, which is designated
as onefold. The transfection efficiency was similar for Neo- and HER-2/neu-transfected
cells, as measured by the percentage of flourescent green cells expressing
GFP (not shown). b, LNCaP/neo cells () or LNCaP/Her-2 cells
() were transfected with PSA P/E-luc and cultured in serum-free media
supplemented with increasing doses of R1881 (left; n = 4 for each)
or R1881 and 5.0 uM casodex (right; n = 3 for each). Luciferase activity
was measured after 48 h. Data are expressed as fold activation relative to
LNCaP/Neo cells in the absence of R1881 or casodex, which is designated as
onefold. Luciferase activation with 1.0 nM R1881 was 49-fold in LNCaP/Neo
cells and 47-fold in LNCaP/Her-2 cells.
HER-2/neu-mediated PSA activation requires androgen receptor To assess the role of AR in this HER-2/neu-mediated signal, we determined
whether the anti-androgen drug casodex, which functions as a competitive inhibitor
for androgen binding to AR, could block the HER-2/neu effect. Casodex inhibited
PSA-P/E-luc activation by R1881 in LNCaP/Neo cells (Fig. 5
b) but had no effect on ligand-independent PSA-P/E-luc activation
by HER-2/neu. The results are consistent with an AR-independent effect of
HER-2/neu or an AR-dependent effect that does not require ligand−receptor
interaction. To distinguish between these, we assessed the effects of HER-2/neu
on PSA-P/E-luc in the hamster kidney epithelial line TS-13 (33) that, unlike LNCaP cells, does not have a highly active
AR pathway. PSA-P/E-luc alone did not function in these cells unless the AR
pathway was reconstituted by the transfection of AR and the addition of R1881
(Fig. 6a). HER-2/neu activated PSA-P/E-luc 19-fold
in TS-13 cells in the absence of added ligand when the cells were co-transfected
with AR. The combination of HER-2/neu, AR and R1881 elicited a 71-fold increase
in PSA-P/E-luc activity compared with a 10-fold increase with AR and R1881.
These results establish that ligand-independent induction of PSA transcription
by HER-2/neu requires a functional AR pathway, and that HER-2/neu and androgen
act synergistically.
Figure 6. Effects of HER-2/neu on androgen receptor function.
a, TS-13 hamster kidney epithelial cells were transfected with PSA
P/E-luc in conjunction with plasmids expressing androgen receptor (AR) and/or
HER-2 in various combinations and in the presence or absence of 1.0 nM R1881
(+ or −). Expression of AR was measured by immunoblot analysis of whole-cell
lysates. Luciferase results are expressed as fold activation relative to TS13
cells transfected with PSA P/E luc in the absence of AR, HER-2/neu and R1881,
which was designated as onefold. b, Similarly, TS-13 cells were transfected
with the PSA E E4-CAT reporter construct in the presence or absence of AR,
HER-2/neu and/or 1.0 nM of R1881. CAT results were analyzed by thin layer
chromatography and quantitated using a phosphorimager. Data are expressed
as fold activation relative to TS13 cells transfected with PSA E E4-CAT in
the absence of AR, HER-2/neu and R1881, which was designated as onefold, and
are the mean (s.d. from three independent experiments. c, Dose−response
of the PSA E E4-CAT reporter to R1881, generated by transfection of TS13 cells
with AR (HER-2/neu with R1881 at concentrations of 0.1 nM, 0.3 nM and 1.0
nM (wedges). CAT activity was measured by thin layer chromatography. At higher
doses of R1881, the PSA E E4-CAT construct was maximally activated and no
additional effect of HER-2/neu was observed (data not shown). d, Dose−response
of a reporter containing a single androgen response element (ARE E4-CAT) to
R1881 with AR (HER-2/neu with R1881 at concentrations of 0.01 nM, 0.1 nM and
1.0 nM (wedges). CAT activity was measured by TLC. The two right lanes contain
higher doses of HER-2/neu plasmid (++).
HER-2/neu 'superactivates' the AR pathway activation The PSA-P/E-luc construct32 encodes 2,900 bp of sequence
containing one well-defined high-affinity AR binding site in the promoter34 and a 496-bp enhancer30. The enhancer encodes a second
high-affinity AR binding site and at least five other non-consensus AR binding
sites, as defined by DNAse I footprinting studies (Y.S. and M.C., unpublished
data). We localized the effect of HER-2/neu on PSA transcription to specific
regions of the promoter/enhancer by constructing two artificial reporters,
one containing the 496-bp enhancer (PSA-E E4-CAT) and a second containing
the high-affinity AR binding site from the PSA promoter (ARE-I E4-CAT). Both
PSA-E E4-CAT and ARE-I E4-CAT were activated in TS-13 cells by R1881 and co-transfection
of AR (Fig. 6b−d). In the presence of AR
, HER-2/neu 'superactivated' PSA-E E4-CAT at three different doses of
R1881, as much as 30-fold above the level seen with AR and R1881 alone (Fig. 6b and c). Using
the ARE-I E4-CAT reporter, transfection of HER-2/neu had no effect beyond
that induced by the combination of R1881 and AR, even at low doses of R1881
and higher doses of HER-2/neu plasmid (Fig. 6d).
Similar results were obtained in LNCaP cells expressing endogenous AR (data
not shown). Thus, the synergistic interaction between HER-2/neu and the AR
pathway can be localized to a 496-bp region of the PSA enhancer, but cannot
be recapitulated using a single high-affinity AR binding site reporter.
Discussion A principal clinical problem in prostate cancer is the conversion of androgen-sensitive
tumors to a hormone-refractory state after treatment with anti-androgen therapy.
The molecular basis for androgen independence is unknown. Here we show that
overexpression of the HER-2/neu receptor tyrosine kinase may be one mechanism.
Increased endogenous HER-2/neu expression is associated with androgen independence
in the LAPC-4 xenograft model, and forced overexpression of HER-2/neu converts
and rogen-dependent LNCaP prostate cancer cells to androgen-independence.
HER-2/neu exerts this effect through modulation of the AR signal transduction
pathway. Specifically, HER-2/neu activates transcription of PSA, an androgen-dependent
serum marker of disease progression that usually correlates with tumor burden
in patients. HER-2/neu and androgen also function synergistically to 'superactivate'
PSA transcription, particularly at low androgen concentrations.
Previous studies of HER-2/neu expression in prostate cancer report conflicting
results. Most groups have focused on radical prostectomy samples, which rarely
contain androgen-independent disease, and report frequencies of HER-2/neu
overexpression that vary widely17,
18,
20,
21,
22,
23. Less
is known about the frequency of HER-2/neu expression in androgen-independent
prostate cancer, mostly because these tumors are rarely biopsied. However,
patients with end-stage, hormone-resistant disease have elevated serum levels
of the Her2/neu extracellular domain24. Future studies using
well-defined HER-2/neu detection reagents are needed to clarify this controversy.
It will also be important to determine if other kinases can activate the
AR pathway and confer androgen-independent growth. The IGF-1 receptor is of
particular interest, as serum IGF-1 levels predict risk of prostate cancer35 and inhibition of the receptor impairs prostate cancer cell growth36. IGF-1, KGF and EGF activate the AR pathway in the absence of ligand10, indicating that the effects of HER-2/neu may not be unique. The
fact that the androgen antagonist casodex can block the effects of IGF-1,
KGF and EGF but not HER-2/neu on AR function may indicate important differences.
The failure of casodex to block PSA induction by HER-2/neu is consistent with
clinical androgen-independent prostate cancer, which arises in patients treated
with anti-androgens, and indicates that HER-2/neu acts on the AR pathway distal
to the interaction between ligand and receptor.
The biochemical details of the cross-talk between the HER-2/neu and AR
pathways are unclear. The fact that HER-2/neu fails to activate a single high
affinity AR binding site supports the idea of the involvement of an accessory
protein, whose function in an AR-dependent transcription complex might not
be measurable on an AR binding site removed from its natural context. Alternatively,
the main effect of HER-2/neu may be to optimize AR function on non-optimal
AR binding sites rather than to enhance AR function on high-affinity binding
sites. HER-2/neu activates the Ras and mitogen-activated protein kinase signaling
pathways37, which may be involved in post-translational modification
of ER (refs. 9,38).
Given the results of studies of nuclear receptors for thyroid hormone, retinoic
acid and others, it is likely that a combination of post-translational modifications
as well as alterations in the assembly of multi-component transcription complexes
may occur39,
40.
Recognizing that there is cross-talk between tyrosine kinase receptor signaling
and the AR pathway has clinical implications. Strategies to inhibit the relevant
tyrosine kinase receptor would be expected to convert androgen-independent
prostate cancer back to a hormone-sensitive state. For HER-2/neu, this might
be tested using a recently developed monoclonal antibody that blocks HER-2/neu
function and has clinical efficacy in breast cancer when combined with chemotherapy41,
42. Alternatively, a detailed understanding of the biochemical
effects of receptor tyrosine kinase signaling on AR function might provide
new drug development insights into targeting the AR pathway downstream of
the point of ligand−receptor interaction.
Methods Cell lines and xenografts. Androgen-dependent and androgen-independent
sublines of the LAPC-4 xenograft were derived as described25.
LNCaP/HER2 and LNCaP/Neo cells were derived by infection with the pLNSXHer2
or pLNSXNeo retrovirus, respectively43, and selection in 500
ug/ml G418. Tumorigenicity was measured by the injection of 1 10
5 cells suspended in 100 ul of Matrigel (Collaborative Biomedical,
Bedford, Massachusetts) subcutaneously into the flanks of intact or castrated
male SCID mice. Tumor size was measured weekly in three dimensions using calipers
as described25. For MTT assays, 1.5 105
LNCaP cells were seeded into 24-well plates in phenol red-free RPMI supplemented
with 10% fetal bovine serum (FBS) overnight. After 12 h, cells were washed,
then re-supplied with phenol red-free RPMI media with complete 10% FBS or
10% FBS that had been treated with charcoal dextran to remove steroid hormone
(Omega Scientific, Tarzana, California). Dihydrotestosterone (Sigma) was added
at defined concentrations. After 48 h, MTT assays were done in triplicate:
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (Sigma) was
added to a final concentration of 5 mg/ml for 4 hours at 37 °C on a multiwell
spectrophotometer, and absorbance was measured at 540 nm.
Protein expression assays. HER-2/neu and PSA expression
were measured by immunoblot using antibody against c-Neu (Calbiochem, La Jolla,
California) at a dilution of 1:1,000 or antibody against PSA (Dako, Carpinteria,
California) at a dilution of 1:250. AR was immunoprecipitated from cell extracts
using anti-AR antibody (PharMingen, San Diego, California) at a dilution of
1:1,000, followed by immunoblot analysis. PSA secretion was quantitated by
ELISA (Hybritech, San Diego, California) of tissue culture supernatant. Equal
cell numbers were plated in phenol red free RPMI containing 10% FBS. Cells
were allowed to attach for 24 h, then the media was changed to serum-free
RPMI with or without 1 nm of the synthetic androgen R1881 (NEN). After another
24 h, 50 l of supernatant was assayed for PSA by ELISA.
Transfections. PSA-P/E-luc (32) was provided by A. Belldegrun (University of California at Los
Angeles). PSA-E E4-CAT was constructed by subcloning the 496-bp enhancer fragment
into E4-CAT (44). ARE-I E4-CAT was constructed
by subcloning a double-stranded oligonucleotide encoding the ARE-I site from
the PSA promoter (AGAACAGCAAGTGCT)(34) into
E4-CAT. LNCaP or LAPC-4 cells were maintained in phenol red-free RPMI supplemented
with 10% FBS. Cells (2 105) were plated in 6-well plates
overnight, then transfected using Tfx-50 (Promega) in 2 ml serum-free OptiMEM
(Life Technologies). TS13 cells were transfected using calcium phosphate.
After 1 h, 2 ml of serum-free media was added, containing varying amounts
of the testosterone analog R1881 without or with 5 uM casodex (Zeneca, Dallas,
Texas). After 48 h, cells were collected in luciferase assay lysis buffer
and analyzed as described (Promega) and normalized to protein content. CAT
assays were done as described45.
Received 25 November 1998; Accepted 21 January 1999
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Acknowledgments We thank J. Redula and C. Tran for assistance with animal experiments,
A. Raitano for advice during the early phases of this work, D. Slamon and
A. Belldegrun for reagents and D. Reese for discussions. This work was supported
by grants from CaP CURE, the Margaret Early Trust and the James S. McDonnell
Foundation and NIH #GM08042.
-Her2)
or the 110-kDa androgen receptor protein (b; 
s.d. from triplicate
wells. c, LN-Neo and LH2-N cells were plated in media supplemented
with either 10% FBS (
) or 10% charcoal-stripped FBS (
), and
MTT assays were done after 48 h. MTT values for charcoal-stripped FBS are
expressed as % relative to complete FBS (representative experiment from seven
independent experiments).
) or sublines that overexpress HER-2/neu (LH2-N, 
;
and LH2-K, 
) were injected into intact (a) and castrated (
b) male mice (n = 8 for each condition). Tumor size was measured
weekly by calipers, and tumors were scored as positive when greater than 0.5
cm in any dimension. Data are presented as % of animals that developed a tumor
versus time. Serum PSA levels were comparable (data not shown).
)
or HER-2/neu (
10
5 cells suspended in 100 ul of Matrigel (Collaborative Biomedical,
Bedford, Massachusetts) subcutaneously into the flanks of intact or castrated
male SCID mice. Tumor size was measured weekly in three dimensions using calipers
as described
l of supernatant was assayed for PSA by ELISA.
