Dehydroepiandrosterone Activates Mutant Androgen Receptors Expressed in the Androgen-Dependent Human Prostate Cancer Xenograft CWR22 and LNCaP Cells
Jiann-an Tan,
Yousuf Sharief,
Katherine G. Hamil,
Christopher W. Gregory,
De-Ying Zang,
Madhabananda Sar,
Paul H. Gumerlock1,
Ralph W. deVere White1,
Thomas G. Pretlow1,
Stephen E. Harris,
Elizabeth M. Wilson,
James L. Mohler1 and
Frank S. French1
Laboratories for Reproductive Biology Departments of Pediatrics
(J.-A.T., K.G.H., C.W.G., D.-Y.Z., M.S., E.M.W., F.S.F.), Biochemistry
and Biophysics (E.M.W.), Surgery (Y.S., J.L.M.), and Pathology (J.L.M.)
and The Lineberger Comprehensive Cancer Center (E.M.W., F.S.F.,
J.L.M.) School of Medicine The University of North Carolina
Chapel Hill, North Carolina 27599 Department of Urology
and Medicine (P.G., R.dW.) University of California Davis Medical
Center Sacramento, California 95817 Department of
Medicine (S.E.H.) The University of Texas Health Science Center
San Antonio, Texas 78284-7756 Department of Pathology
(T.G.P.) School of Medicine Case Western Reserve University
Cleveland, Ohio 44160
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ABSTRACT
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An androgen receptor (AR) gene mutation
identified in the androgen-dependent human prostate cancer xenograft,
CWR22, changed codon 874 in the ligand-binding domain (exon H) from CAT
for histidine to TAT for tyrosine and abolished a restriction site for
the endonuclease SfaNI. SfaNI digestion of AR exon H DNA
from normal but not from prostate cancer tissue indicated H874Y is a
somatic mutation that occurred before the initial tumor transplant.
CWR22, an epithelial cell tumor, expresses a 9.6-kb AR mRNA similar in
size to the AR mRNA in human benign prostatic hyperplasia. AR protein
is present in cell nuclei by immunostaining as in other
androgen-responsive tissues. Transcriptional activity of recombinant
H874Y transiently expressed in CV1 cells in the presence of
testosterone or dihydrotestosterone was similar to that of wild type
AR. With dihydrotestosterone at a near physiological concentration
(0.01 nM), H874Y and wild type AR induced
2-fold greater luciferase activity than did the LNCaP mutant AR
T877A. The adrenal androgen, dehydroepiandrosterone (10 and 100
nM) with H874Y stimulated a 3- to 8-fold
greater response than with wild type AR and at 100
nM the response was similar with the LNCaP
mutant. H874Y, like the LNCaP cell mutant, was more responsive to
estradiol and progesterone than was wild type AR. The antiandrogen
hydroxyflutamide (10 nM) had greater agonist
activity (4- to 7-fold) with both mutant ARs than with wild type AR. AR
mutations that alter ligand specificity may influence tumor progression
subsequent to androgen withdrawal by making the AR more responsive to
adrenal androgens or antiandrogens.
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INTRODUCTION
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Human prostate cancer is androgen dependent initially, but after
androgen withdrawal therapy it acquires the ability to grow in the
absence of testicular androgens. Androgen receptor (AR) is expressed in
the majority of untreated prostate cancers and in most that recur
during androgen withdrawal therapy. This continued presence of AR
suggests that androgen responsiveness may persist in tumors that recur
during therapy. Major goals of current research on prostate cancer
include identifying mechanisms that stimulate recurrent growth. One
mechanism could involve mutations that alter the normal AR
ligand-binding specificity, thereby enabling AR activation by adrenal
androgens or antiandrogens used in cancer treatment. Other possible
mechanisms include ligand-independent activation of AR and adaptation
to alternate growth regulators.
The first AR missense mutation identified in human prostate cancer was
in the LNCaP cell line (1, 2, 3, 4). Subsequently, amino acid substitutions
were reported in organ-confined as well as metastatic tumors (5, 6, 7, 8, 9, 10, 11).
Of the several AR mutants in prostate cancer that have been
characterized thus far, the majority are functional and can mediate
androgen-induced transactivation, in contrast to loss of function
germ-line AR mutations that cause androgen insensitivity.
CWR22 is an androgen-dependent human prostate cancer xenograft
propagated in male nude mice (12, 13). Upon androgen withdrawal, CWR22
prostate-specific antigen mRNA and protein decrease rapidly, cells
undergo apoptosis (13, 14), and tumors regress in size; but after
several months they recur in the absence of testicular androgens in a
manner characteristic of human prostate cancers (13, 14). In this
report a mutant AR with altered ligand specificity is identified and
shown to be expressed in CWR22 epithelial cells. Ligand-dependent
activation of the mutant AR in transient cotransfection assays is
compared with that of the mutant AR in LNCaP cells. The CWR22 AR mutant
is transcriptionally active in response to testicular androgen like
wild type AR but differs from wild type in that it is also activated by
the adrenal androgen, dehydroepiandrosterone, the antiandrogen,
hydroxyflutamide, and by estradiol and progesterone.
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RESULTS
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Localization of AR in the CWR22 Human Prostate Cancer Xenograft
The majority of nuclei in CWR-22 stained positively with the AR
antibody AR-52 (15), some more intensely than others (Fig. 1B
). A preimmune control panel (Fig. 1C
) indicated that
staining in Fig. 1B
is AR specific. Hematoxylin-eosin staining (Fig. 1A
) illustrates the predominance of epithelial cells with lack of
surrounding stroma. Northern blot analysis of CWR22 total RNA revealed
a 9.6-kb AR mRNA (Fig. 2A
) similar in size to the AR
mRNA expressed in benign prostatic hyperplasia tissue (15). In an
RT-PCR assay, expression of AR and prostate-specific antigen (PSA)
mRNAs in CWR22 was similar to that in the androgen-responsive LNCaP
cell line (Fig. 2B
) and contrasted with the undetectable or very low
levels in the androgen- unresponsive cell lines, DU145, PC3, and
TSU-PR1. These results are consistent with the androgen-responsive and
relatively differentiated state of CWR22. In mice bearing CWR22
xenografts, Wainstein et al. (13) observed that serum PSA
concentrations correlate with tumor size, and blood levels of PSA
decrease after androgen withdrawal. More recently, Gregory et
al. (14) demonstrated a decrease in CWR22 PSA mRNA levels within
48 h after androgen withdrawal.

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Figure 1. Immunohistochemical Analysis of AR Protein
Expressed in Androgen- Stimulated Epithelial Cells of CWR22 Tumor from
an Intact Nude Mouse
Frozen tissue sections were fixed and immunostained with protein
A-purified AR antibody, AR-52. A, Hematoxylin-eosin; B, AR-52; C,
peptide-absorbed AR-52 control.
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Figure 2. Expression of AR mRNA in the Androgen-Dependent
Human Prostate Cancer Xenograft, CWR22, and the Androgen-Responsive
LNCaP Cell Line
A, Northern hybridization of CWR22 total RNA from an
androgen-stimulated nude mouse using a human AR cDNA probe. The major
AR mRNA species is 9.6 kb. B, Southern hybridization of AR and PSA,
RT-PCR products from LNCaP, DU145, PC-3, and TSU-PR1 cells and CWR22.
DU145, PC-3, and TSU-PR1 are androgen-unresponsive human prostate
cancer cell lines. Although not apparent in this figure, PC3 cells
contained a very low level of AR mRNA.
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Identification of the AR Gene Mutation in CWR22
To search for the presence of an AR gene mutation, DNA from
CWR22 was PCR amplified using sets of intron primers that bracket each
exon (16, 17). Exon A was amplified using four overlapping sets of
primers. All exons amplified to the correct size, indicating no major
deletions. Analysis of AR exon H by denaturing gradient gel
electrophoresis revealed extra doublet bands (Fig. 3B
),
suggesting a mismatch in the heteroduplex of wild type and CWR22 DNA.
No similar indication of an AR gene mutation was detected in exons A
through G. Automated sequencing of exon H DNA detected a C to T
mutation at position 4046, numbered according to Lubahn et
al. (16), that changed codon 874 from CAT coding for histidine to
TAT coding for tyrosine (Fig. 3
, A and C). The mutation was detected
also by RT-PCR from CWR22 total RNA and manual sequencing of the cDNA
region encoded by exon H. Substitution of T for C 4048 altered a
SfaNI recognition sequence (GCATC to GTATC).
SfaNI did not cut exon H DNA from the original CWR22
xenograft, indicating that the AR mutation was likely present in the
tumor before transplantation. In contrast, SfaNI cut AR exon
H DNA amplified from paraffin-embedded normal testicular tissue of the
same patient, indicating absence of the C to T mutation (data not
shown). Thus, H874Y was a somatic mutation in the prostate tumor not
present in germline DNA.

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Figure 3. A Single Base Substitution C to T Changed His 874
to Tyr (H874Y) in the CWR22 AR
A, Schematic showing location of the missense mutation in exon H of the
steroid-building domain. B, Denaturing gradient gel electrophoresis of
exon H PCR-amplified DNA from wild type and CWR22 AR genes. C,
Nucleotide sequence of the portion of CWR22 AR exon H containing a
T substituted for the wild type C. The four nucleotides
represented by solid, broken, and dashed
lines are color coded in the original tracing.
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Steroid-Dependent Transactivation of the CWR22 AR Mutant in
Comparison with the LNCaP Cell Mutant AR
Since AR mutation H874Y in CWR22 tumor cells is located near AR
mutation T877A of the LNCaP cell line, we compared the transcriptional
activities of these mutants in response to a variety of ligands
including adrenal and sex steroids and the antiandrogen,
hydroxyflutamide. The mutant AR in LNCaP cells was identified earlier
by cloning the AR cDNA from an LNCaP cell cDNA library in the
-ZAPII
cloning vector (2, 3). AR mutations were constructed in the expression
vector, pCMVhAR (18, 19), and cotransfected into CV1 cells together
with a mouse mammary tumor virus long terminal repeat-luciferase
reporter vector.
CWR22 AR mutant H874Y induced higher luciferase activity than wild type
AR (P < 0.01) at 0.01 nM testosterone (T),
a concentration close to the physiological range of free T in human
blood (20) (Fig. 4
). The response of H874Y to
dihydrotestosterone (DHT) at 0.01 nM was similar to that of
wild type AR and about 2-fold more active (P < 0.001)
than LNCaP mutant T877A. Activity induced by 1 nM
androstenedione was similar with H874Y or wild type AR and slightly
higher (P = 0.001) than T877A (Fig. 4
). Little
transactivation was observed at lower concentrations of
androstenedione. Because androstenedione has low affinity for the wild
type AR (6), its induction of transcriptional activity with both mutant
and wild type ARs likely resulted from conversion to T and DHT in CV1
cells. Relative responses of wild type and mutant ARs to T and DHT and
to androstenedione were similar.

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Figure 4. Ligand-Dependent Transactivation by CWR22 (H874Y)
and LNCaP (T877A) Mutants with T, DHT, and Androstenedione (ASD)
Transient cotransfection assays were performed in CV1 cells using a
mouse mammary tumor virus long terminal repeat-luciferase reporter gene
(2.5 µg) together with the full-length wild type human AR, pCMVhAR,
or mutant AR expression vector (0.1 µg). Cells were incubated 40
h in the presence and absence of steroid at the concentrations
indicated. Cells were lysed and luciferase activity measured in a
luminometer as described in Materials and Methods. Data
are plotted as means ± SEM.
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The adrenal androgen dehydroepiandrosterone (DHEA) at 1 nM
stimulated luciferase activity 2-fold with mutant and wild type ARs. At
10 nM DHEA, H874Y transcriptional activity was 3-fold
greater (P < 0.001) than that of wild type AR and
increased to about 8-fold greater (P < 0.001) at 100
nM DHEA (Fig. 5
). At 100 nM
DHEA, transcriptional activity with H874Y was similar to that of T877A.
DHEA had little agonist activity with wild type AR up to 100
nM, indicating there was minimal conversion of DHEA to T or
DHT in CV1 cells. This concentration of DHEA is similar to that
reported in human prostate tissue as discussed below.

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Figure 5. Transactivation by CWR22 (H874Y) and LNCaP (T877A)
Mutant and Wild Type AR with the Adrenal Androgen DHEA
Transient cotransfection assays were performed in CV1 cells as
described in Fig. 4 and Materials and Methods with
concentrations of DHEA as indicated.
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Estradiol at 0.1 nM stimulated 2-fold greater
(P < 0.001) luciferase activity with H874Y than wild
type AR, and this difference increased to several fold
(P < 0.001) at 1 nM estradiol (Fig. 6
). Induction of transcription with T877A was also
greater (P < 0.001) than with wild type AR at 1
nM estradiol but was only about half that of H874Y. The
physiological level of total serum estradiol in the adult male is
approximately 0.1 nM whereas free estradiol is 12% of
the total (20). There was little increase in transcription at 0.01
nM estradiol suggesting H874Y would not be activated in the
adult male; however, aromatase activity in human prostate may cause
tissue levels to be higher than in serum. The CWR22 mutant AR
transcriptional response to 1 nM progesterone was greater
(P < 0.01) than that of the wild type AR but less
(P < 0.01) than the LNCaP mutant (Fig. 6
). Total serum
progesterone concentration in adult males,
1 nM, is
similar to that in females during the follicular phase of the menstrual
cycle (20) and may be sufficient to activate a mutant AR in
prostate.

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Figure 6. Transactivation by CWR22 (H874Y) and LNCaP (T877A)
Mutant and Wild Type AR in the Presence and Absence of Estradiol
(E2) or Progesterone at the Concentrations Indicated
Transient cotransfection assays were performed as described in Fig. 4
and Materials and Methods.
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The antiandrogen hydroxyflutamide had low agonist activity with mutant
and wild type ARs at concentrations of 1 nM or less (Fig. 7
). However, at 10 nM hydroxyflutamide,
H874Y and T877A transcriptional activities were 4- and 6-fold greater
(P < 0.001) than that of wild type AR (Fig. 6
).
Maximal luciferase activities stimulated by DHEA and hydroxyflutamide
with the mutant ARs were 1520% of activities induced by T and
DHT.

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Figure 7. Transactivation by CWR22 (H874Y) and LNCaP (T877A)
Mutant and Wild Type AR in the Presence and Absence of the Antiandrogen
Hydroxyflutamide (OH-FL) at the Concentrations Indicated
Transient cotransfection assays were performed in CV1 cells as
described in Fig. 4 and Materials and Methods.
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DISCUSSION
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CWR22 is an androgen-dependent human prostate cancer epithelial
cell xenograft lacking in stroma cells. We have shown that CWR22
expresses a mutant AR, H874Y, with altered ligand responsiveness.
Absence of SfaNI digestion of PCR-amplified exon H DNA
indicated CWR22 tumor cells express only the mutant AR. The CWR22
mutant AR mediated a transcriptional response to T and DHT similar to
that of wild type AR. DHEA, estradiol, progesterone, and
hydroxyflutamide induced greater transcriptional responses with the
H874Y mutant than with wild type AR. H874Y-mediated transcriptional
responses to DHEA and estradiol were greater whereas responses to
progesterone and hydroxyflutamide were less than those of the LNCaP
mutant T877A.
DHEA-induced transactivation by H874Y and T877A was highest with 100
nM DHEA, a concentration likely maintained in human
prostate tissue by adrenal secretion of DHEA and DHEA-sulfate. Total
serum DHEA in adult males (mean,
25 nM) is within the
range of total serum testosterone. However, relatively more serum DHEA
should be available to prostate cells because it circulates loosely
bound to albumin (20). Serum DHEA-sulfate can be 300500 times the
concentration of DHEA, and the sulfatase present in human prostate
converts DHEA-sulfate to DHEA (21). Mean concentrations of DHEA-sulfate
and DHEA in nonhyperplastic tissue specimens obtained by total
prostatectomy were
300 and 90 pmol/mg DNA, respectively (equivalent
to 300 and 90 nM, assuming
1 mg DNA/g tissue) (21). In a
patient undergoing androgen withdrawal therapy by castration or LH
suppression, DHEA could be in a concentration range sufficient to
activate mutant ARs such as H874Y, T877A, or the AR mutant V715M
identified in a prostate cancer bone marrow metastasis by Culig
et al. (6). In human prostate, low levels of
4,5 isomerase activity limit DHEA conversion to
4 androgens (21). However, small amounts of DHT were
formed from DHEA and DHEA-sulfate in human benign prostatic hyperplasia
tissue (22).
Estradiol derived from peripheral conversion of adrenal androstenedione
is a potential agonist for mutant ARs in prostate cancer. Estradiol
induced an increase in luciferase activity with both the CWR22 and
LNCaP mutants at a concentration of 10-9 M,
less than the amount required for half-maximal stimulation of LNCaP
cell growth (10-8 M). In our transcription
assays, estradiol was a greater agonist with the CWR22 mutant AR than
with the LNCaP mutant. However, CWR22 does not grow in female nude
mice, indicating growth stimulation by H874Y is not activated by female
mouse levels of estradiol. It was assumed the LNCaP mutant AR mediates
the growth-stimulating effect of estradiol on LNCaP cells (2, 3, 4).
However, a recent report demonstrated that LNCaP cells also contain
estrogen receptors based on ligand binding and immunocytochemical
assays (23). The antiestrogen ICI-182,780 inhibited estradiol
stimulation of growth but did not inhibit growth induced by DHT,
suggesting the growth effect of estradiol was mediated by estrogen
receptors rather than by the T877A mutant AR. It remains to be
determined whether higher concentrations of estradiol stimulate CWR22
tumor growth by way of the mutant AR. Estrogen receptors were not
detected by immunostaining CWR22 tumor cells with three different
antibodies to the human estrogen receptor, a finding in agreement with
other immunohistochemical studies showing weak or undetectable staining
of estrogen receptors in benign prostatic hyperplasia or prostate
cancer epithelial cells (24). It is not yet established whether the
human homolog of rat prostate estrogen receptor ß (25) is expressed
in CWR22.
Hydroxyflutamide is the active metabolite of flutamide, an androgen
antagonist used in the treatment of prostate cancer. Both H874Y and
T877A mediated increased transcriptional activity at 10 nM
hydroxyflutamide whereas there was no effect with wild type AR. Another
AR mutant in human prostate cancer, V715M, was unresponsive to 100
nM hydroxyflutamide (6) but responded 2-fold greater than
wild type AR to micromolar concentrations as did the prostate cancer AR
mutant V730M (26). In the absence of androgen, hydroxyflutamide is an
agonist with wild type AR at concentrations of 1 µM or
higher (27, 28). As noted by Wong et al. (27) and by
Kemppainen and Wilson (29), this could be of clinical importance in the
regression of prostate cancer observed in some patients whose flutamide
treatment has been withdrawn (30, 31). Prostate cancer patients treated
with high-dose flutamide can have plasma levels of hydroxyflutamide
greater than 1 µM. During androgen withdrawal therapy,
hydroxyflutamide could function as an agonist when concentrations
greatly exceed the concentration of androgen, although its affinity for
wild type AR is at least 1 order of magnitude less than that of DHT (6, 27). AR mutants such as H874Y and T877A with enhanced responsiveness to
hydroxyflutamide would be more active than wild type AR in mediating
growth effects of hydroxyflutamide on prostate cancer cells.
Half-maximal stimulation of LNCaP cell growth in culture was achieved
with 10-8 M hydroxyflutamide (data not shown),
in agreement with other studies (32, 33, 34). In general, the relative
growth-stimulating activity of an AR agonist correlates with its
binding affinity for AR and induction of AR-mediated transactivation in
transient cotransfection assays (2, 3, 4, 32, 33, 34, 35, 36, 37).
Of the different AR missense mutations reported in clinical prostate
cancer (reviewed in Refs. 11 and 38 and B. Gottlieb, Androgen Receptor
Gene Mutations Database), most that were characterized functionally
retain androgen-dependent transcriptional activity. V730M AR from a
stage B, organ-confined prostate cancer had no loss of androgen binding
or transcriptional activity (5, 39). The LNCaP cell line containing AR
mutant T877A was derived from a lymph node metastasis. Three additional
mutations, V715M, T877S, and H874Y, were identified in bone metastases
(6, 7). Each of the mutants exhibited essentially normal
transactivation in response to DHT. The V715M mutant showed no steroid
binding abnormality but had increased transcriptional activity relative
to wild type AR in response to the adrenal androgens, DHEA and
androstenedione (6). Mutants H874Y and T877S were reported to have
enhanced transcriptional activity with progesterone and estradiol at
concentrations of 10 and 100 nM (7). Mutant Q919R at the
carboxyl terminus of AR remained functional in response to DHT, with
only partial loss of activity (40). The only loss of function mutations
reported in association with prostate cancer are a single base change
that introduced a stop codon (Trp 794 to stop) in the steroid-binding
domain (9) and C619Y in exon C close to the DNA-binding domain (40). By
analogy with the crystal structures of the thyroid hormone receptor and
retinoic acid receptor
ligand-binding domains (41, 42), AR ligands
are buried in a hydrophobic pocket formed by several
-helices folded
in a way such that widely separated amino acids are brought together to
form the pocket. AR amino acids 874 and 877 are located within a
conserved heptad repeat of hydrophobic residues (38, 43) in
-helix
11 (41).
Since AR is required for androgen-dependent growth of prostate cancer
cells, tumors may select for AR mutants that retain activity. Most
prostate cancers that relapse during androgen withdrawal therapy
express AR at levels similar to androgen-dependent prostate cancer
(Refs. 44 and 45 and J. L. Mohler, unpublished). Functional AR mutants
with enhanced responsiveness to adrenal androgens and/or antiandrogens
could have an active role in the survival and recurrent growth of
prostate cancer cells. Moreover, the altered spectrum of ligand
responsiveness may potentiate ligand-independent activation of AR in
relapsed prostate cancers with increased kinase activities
(46, 47, 48).
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MATERIALS AND METHODS
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Monkey kidney CV1 cells were obtained from the American Type
Culture Collection. DMEM/high glucose and DMEM:F12 were from GIBCO
(Grand Island, NY), and FCS was purchased from Hyclone Laboratories,
Inc. (Logan, UT). CV1 cell lysis buffer was supplied by Ligand
Pharmaceuticals Inc. (San Diego, CA); D-luciferin was from
Analytical Luminescence (Westlake Village, CA). Hydroxyflutamide was
provided by R. O. Neri Schering Corp. (Bloomfield, NJ); other steroids,
buffers, and chemicals were from Sigma (St. Louis, MO), E.M. Science
(Fort Washington, PA) and Fisher (Pittsburgh, PA). Taq
polymerase and AMV reverse transcription were from Promega (Madison,
WI), and Deep Vent DNA polymerase and SFaNI were from New
England Biolabs (Beverly, MA).
Human Prostate Cancer Tissue (CWR22)
CWR22 is an androgen-dependent, human prostatic carcinoma
xenograft established from a transurethral resection and grown
subcutaneously in male athymic nude mice, each containing a 12.5-mg
testosterone pellet (Innovative Research, Sarasota, FL) implanted
subcutaneously (12). The tumor was stage D with osseous metastases,
Gleason grade 9. After removal of CWR22 xenografts from nude mice,
tumor tissue was frozen immediately in liquid nitrogen and stored at
-80 C.
Immunocytochemistry
Small pieces of fresh tumor tissue (
1 cm2 x 0.3
cm thick) were frozen in isopentane precooled in liquid nitrogen on
brass mounts using minced rat liver cushions as an adhesive. Tissue was
stored in liquid nitrogen until sectioned at 6 µm thickness using a
cryostat at -31 C. Preparation of antibody AR-52 and the method for
immunostaining were as described previously (15, 49, 50).
Nucleic Acid Extraction and Northern Hybridization of Human
AR
Frozen CWR22 tumor tissue was thawed, finely minced with
scalpels, digested with proteinase K at 37 C overnight, and DNA
extracted with phenol-chloroform. RNA was removed by RNase digestion
and phenol-chloroform extraction. For extraction of normal testicular
tissue DNA from the same patient, 6 µm thick sections were cut from
paraffin blocks and deparaffinized in xylene. Tissue was digested with
proteinase K, and DNA was isolated by NaCl deproteinization (10). Total
RNA was extracted by a single-step method using acid-guanidinium
thiocyanate-phenol-chloroform (51). Northern hybridization of AR mRNA
was as described by Quarmby et al. (52).
Cell Lines
The human prostate tumor cell lines LNCaP, DU145, and PC-3 were
obtained from the American Type Culture Collection (Rockville, MD) and
maintained according to their instructions. The TSU-Pr1 cell line was
generously provided by Dr. Edward Gelmann (Georgetown University School
of Medicine, Washington DC) and maintained in DMEM supplemented with
10% FBS. Cultures were harvested for RNA analysis at approximately
80% confluence.
RT-PCR Analysis of Human AR and PSA mRNA
RT-PCR analysis of AR mRNA was done essentially as
previously described (45). Briefly, total RNA was extracted from the
cell lines and xenografts using guanidinium isothiocyanate and CsCl
density gradient centrifugation. Total RNA was transcribed to cDNA
using RT primed with random hexamers. Complementary DNA equivalent to
25 ng of the initial total RNA was subjected to RT-PCR using the
following oligonucleotides as primers for the AR
transcripts: PG45 (upstream, sense) 5'-CCTGATCTGTGGAGATGAAGCTTC-3' and
PG46 (downstream, antisense) 5'-TGTCGTGTCCAGCACACACTACAC-3'. These
primers are located in exons B and D, respectively, and create a 495-bp
PCR product from cDNA. The oligonucleotide PG42
(5'-TGGGAGCCCGGAAGCTGAAGAAAC-3') (spanning the junction of exons C and
D) is internally positioned to the two primers and served as the probe
for detecting the products in Southern blotting. PCR was run for 36
cycles under the conditions of 95 C (30 sec), 60 C (30 sec), and 72 C
(30 sec). The 36 cycles were followed by a 10-min extension phase at 72
C. Southern blotting of the PCR products was done as previously
reported using a nonisotopic enzyme chemiluminescent detection system
(53). For analysis of expression of PSA transcripts, the primers
PSA-494 (upstream, sense) 5'-TACCCACTGCATCAGGAACA-3' and PSA-894
(downstream, antisense) 5'-GTCCAGCGTCCAGCACACAG-3' were used to create
a 421-bp product (54). PCR reaction parameters were 94 C for 15 sec, 60
C for 15 sec, and 72 C for 45 sec for 35 cycles followed by a 10-min
extension at 72 C. The oligonucleotide used as the probe for Southern
blotting of PSA RT-PCR products was PSA-559
5'-ACACAGGCCAGGTATTTCAG-3'. Sample loadings were adjusted to equivalent
levels of c-N-ras as previously described and validated by Fishman
et al. (55)
Analysis of the AR Gene in CWR22
Amplification of AR gene DNA.
Exons B through H were amplified individually using GC-clamped intron
primers. Due to its large size, exon A was amplified in four fragments
using exon primers (16, 17). PCR was performed for 30 cycles of
denaturation (95 C for 1 min), annealing (60 C for 1 min), and
extension (72 C for 2 min). In each PCR a negative control containing
no DNA was included. PCR product size was verified by electrophoresis
on 1% agarose gels in Tris-borate buffer containing 2 mM
EDTA, 50 mM Tris base, 50 mM boric acid, pH 8.0
(TBE), and ethidium bromide staining.
Denaturing Gradient Gel Electrophoresis.
Electrophore-sis of amplified AR was performed on denaturing
gradient gels as described (17). Heteroduplexes for each exon were
formed by mixing equal amounts of test sample and wild type PCR
products. DNA was denatured at 95 C and reannealed by slow cooling to
room temperature.
RT-PCR for Splicing Errors.
Total RNA (1 µg) was reverse transcribed into cDNA using AMV RT and
oligo (dT)20 primer. Primers for PCR were chosen to amplify
all cDNA splice sites from exons A through H, a region of 1296 bp.
Amplified AR cDNA fragments were electrophoresed in 1% agarose gels in
TBE and stained with ethidium bromide.
Restriction Site Analysis.
Approximately 200 ng of PCR product of exon H amplification were
incubated with 2 U of the restriction enzyme SfaNI in a
total volume of 20 µl of NE Buffer 3 for 16 h as recommended by
New England Biolabs (Beverley, MA). After incubation, the enzyme was
heat inactivated and the DNA analyzed by electrophoresis in a 1%
agarose gel in TBE buffer.
Subcloning and Sequencing the CWR22 Mutation
An aliquot of the PCR reaction used in denaturing gradient gel
electrophoresis was chloroform extracted and ethanol precipitated. The
DNA ends were made blunt with Klenow fragment (Promega, Madison WI) and
phosphorylated with T4 kinase (Promega). These fragments were cloned
into pBluescript II SK- (Stratagene, La Jolla, CA) cut with
EcoRV (Promega), and dephosphorylated with calf intestinal
phosphatase (Boehringer Mannheim, Indianapolis, IN).
DNA sequence was obtained from four individual clones using
double-stranded sequencing with Taq Dideoxy Terminator and
an Applied Biosystems 373A automated DNA sequencer (Foster City, CA).
Standard M13 vector forward and reverse primers were used. Sequence for
both strands of each clone was analyzed using a Genetics Computer Group
(GCG) program (Madison, WI).
Construction of Mutant AR Expression Vectors
The CWR22 mutant AR H874Y vector was constructed in pCMVhAR
using the two-step PCR method as described (17, 18). LNCaP AR was
cloned from an LNCaP cDNA library in
Zap-II (2, 3). The LNCaP
mutant AR T877A cDNA (2) HindIII/BamHI fragment
replaced the wild type HindIII/BamHI fragment in
pCMVhAR. Expression vector constructs were analyzed by DNA
sequencing.
Transcription Assay
Transcriptional activities of wild type and mutant ARs were
analyzed by transient cotransfection of African green monkey CV1 cells
using a mouse mammary tumor virus long terminal repeat-luciferase
reporter vector as described (27). CV1 cells were maintained at 37 C
under 5% CO2 in high glucose DMEM with 4.5 g/liter glucose
supplemented with 5% FBS. On the day before transfection, 4.5 x
105 cells per 6-cm culture dish were grown in the same
medium for about 20 h until 7080% confluent. Expression vector
DNA (100 ng) and reporter vector DNA (2.5 µg) were transfected into
CV1 cells using CaPO4 DNA precipitation (56). Cells were
incubated 40 h in serum-free medium with and without steroids with
a change in medium at 24 h and harvested on the plate using lysis
buffer (Ligand Pharmaceuticals Inc., San Diego, CA). The luciferase
assay was performed as described (27). Background activity was defined
as light units determined in the presence of the AR expression vector
and the absence of ligand. Stimulation of luciferase activity is
expressed as fold increase over background based on five or more
independent experiments. Background activities of the AR expression
vectors in light units were: wild type hAR 1817 ± 521, H874Y
2904 ± 1039, and T877A 3718 ± 2153. Students t
test and the computer program Sigmastat were used for statistical
analysis. Data are expressed as the means ± SEM of
three to 11 experiments.
 |
ACKNOWLEDGMENTS
|
---|
We thank K. Michelle Cobb and Yeqing Chen for technical
assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Frank S. French, M.D., Laboratories for Reproductive Biology, University of North Carolina, School of Medicine/CB 7500, Chapel Hill, NC 27599-7500.
This work was supported by NIH Grants AG-11343, HD-04466, P30-HD-18968
(DNA, Cell Culture and Histochemistry Cores), T32-HD-07315 (J-A.T. and
C.W.G.), CA-57179 (T.G.P.) and CA-55792, CA-55792 (R.W. de V.W.).
1 Cooperative Network for Molecular and Genetic Markers for Prostate
Cancer, National Cancer Institute. 
Received for publication September 3, 1996.
Revision received December 16, 1996.
Accepted for publication January 15, 1997.
 |
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