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Departments of Urology (S.Z., M.-L.H., W.Z., D.J.T., C.Y.F.Y.) and Biochemistry and Molecular Biology (D.J.T., C.Y.F.Y.), Laboratory Medicine and Pathology (G.G.K.), Mayo Graduate School, Mayo Clinic/Foundation, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Charles Y. F. Young, Mayo Clinic/Foundation, 200 First Street, Southwest, Rochester, Minnesota 55905.
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Abstract |
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Using gene transfer assays, T3 alone showed no effect on transcriptional activation of a reporter gene mediated by the PSA or hK2 enhancer/promoters. T3 potentiated the androgen-mediated transcription of the PSA gene but not that of the hK2 gene. A previous study suggested that the T3 effect on PSA protein expression was caused by an up-regulation of the androgen receptor (AR) protein by T3. Our results contradict these. Although AR expression was increased by T3 alone, Western blot analysis showed that the total cellular AR level was not further increased by T3 in the presence of androgens, in comparison with cells stimulated by androgens alone. Both Western blot analysis and a gel DNA band shift assay revealed that nuclear AR was not increased by T3. This study suggests that transcription factor(s) other than the AR may mediate T3 enhancement of androgenic induction of PSA expression.
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Introduction |
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The LNCaP cell line, which was derived from a human prostate cancer lymph node metastasis, is well differentiated and androgen responsive. It still exhibits many phenotypic features similar to that of normal prostate epithelia. For example, LNCaP cells express androgen, thyroid hormone, and several other nuclear receptors, as well as a number of tissue-specific differentiation markers, such as prostate-specific antigen (PSA), human glandular kallikrein (hK2), and prostatic acid phosphatase (7, 8, 9, 10). The androgen receptor (AR) in LNCaP cells contains a single point mutation, which alters its steroid specificity but otherwise functions normally (11, 12). However, in response to androgen deprivation, LNCaP cells exhibit a low level of in vitro apoptosis and a partial in vivo apoptosis (13). In this paper, we used LNCaP cells to study the interactive effects of T3 and androgens on the regulation of the prostate-specific differentiation marker genes, PSA and hK2. We also studied the effect of T3 and androgens on proliferation of prostate cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For PSA and hK2 expression, LNCaP cells were seeded at 4 x 104/ml·well in 24-well dishes and treated with different concentrations of T3, either with or without 1 nM Mib. After a 7-day incubation, spent media were harvested, and PSA and hK2 were quantified by an immunometric assay, as described below. Cell density was quantified by a cell proliferation assay described below. The concentrations of PSA and hK2 were normalized by cell density measurements and expressed as ng/ml·A490 (A490 is absorbance at 490 nm).
Cell proliferation assays
Cell proliferation was measured with a nonradioactive assay.
Solutions containing the tetrazolium compound
[3-(4,5-dimethylthiazol)-2-yl-5-(3-carboxymeth-oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt (MTS)] and an electron coupling reagent (phenazine
methosulfate) were used in the assay (14). MTS is bioreduced by
metabolically active cells into a formazan that is soluble in tissue
culture medium. The absorbance of the formazan product was read at 490
nm. This absorbance is directly proportional to the number of living
cells in culture (data not shown). All reagents were purchased from
Promega Corp. (Madison, WI). Cells were incubated with
freshly prepared, combined MTS/phenazine methosulfate (ratio of 1:1 by
volume) solution for 1.5 h at 37 C in a humidified 5%
CO2 atmosphere.
Immunoassays for PSA and hK2 quantitation
Monoclonal antibodies (mAbs) against hK2 were developed in
collaboration with Hybritech Inc. (San Diego, CA) (15, 16, 17). PSA levels
in culture supernatant were determined by an immunoenzymatic assay
using the Tandem-E PSA kit (Hybritech Inc.). Levels of hK2 were
measured by an immunometric sequential (sandwich) assay with mAbs.
Details have been described previously (17). Briefly, the capture
antibody was coated on quarter-inch polystyrene beads (Clifton
Plastics, Ltd., Fort Erie, Ontario, Canada) at 1 µg of
antibody per bead, and the detection antibody was prelabeled with
acridinium ester. Recombinant hK2 (2,500 ng/ml, Hybritech Inc.) was
serially diluted into assay diluent and used as an antigen in the
standard curve. The capture antibody on the beads was incubated with
the analyte in the sample or standard hK2 solution for 2 h at 37
C. Unbound antigen was washed away. The detecting antibody, labeled
with acridinium ester, was added to the bead and incubated for 2 h
at 37 C. Unreacted, excess antibody was removed by washing, leaving the
antigen sandwiched between the two mAbs on the beads. The
chemiluminescent signal (expressed as relative light units) was
detected in a Magic Lite Analyzer II (Chiron Diagnostic Corp., Norwood, MA). Concentrations of hK2 were calculated from
the standard curve derived from linear regression of the relative light
units data.
Transient transfection assay
LNCaP cells were grown under the same conditions as described
above. After depletion of steroids, cells were transfected with pBLCAT3
containing either the PSA enhancer/promoter (PSA E-407), as reported
previously (17), or the hK2 5-kb promoter, using liposomes containing
DDAB (dimethyldioctadecyl-ammonium bromide) and L-a-lecithin (4:10)
under serum-free conditions. The ß-gal-CMV vector (pCMVB,
CLONTECH Laboratories, Inc., Palo Alto, CA) was
cotransfected to normalize for transfection efficiency. The parental
vector pBLCAT3 was used as a control. After transfection, cells were
treated with Mib, T3, both, or vehicle for 24 h in 5%
charcoal-stripped FBS-RPMI 1640 culture medium. Charcoal was used to
remove the endogenous steroids from the serum. Cell extracts were
prepared from duplicate plates and used for chloramphenicol
acetyltransferase (CAT) and ß-galactosidase assays, according to a
published method (18). Three independent transfections were
performed.
Nuclear extracts
LNCaP cells were grown under the same conditions as described
above, except that, before nuclear extraction, the cells were treated
with or without 1 nM Mib ± 10-7
M T3 in the presence of 5% charcoal-stripped
FBS for 24 h. Nuclear extracts were prepared as described (19).
Briefly, cells were collected with Ca2+,
Mg2+–free PBS containing 1 mM EDTA and were
centrifuged at 1,000 rpm at 4 C for 10 min in a JA 68 rotor
(Beckman Coulter, Inc., Fullerton, CA). The cell
pellet was washed with ice-cold PBS, resuspended in 10 ml buffer
containing 15 mM Tris-HCl (pH 7.5), 1 mM EDTA,
10% glycerol, and 2 mM dithiothreitol (DTT) and was
centrifuged for 10 min at 5,000 rpm in a JA20 rotor from Beckman Coulter, Inc. The pellet was resuspended in the same buffer (3
ml), homogenized with 20 strokes of a B pestle in a dounce homogenizer,
and centrifuged at the same speed to remove the supernatant. The crude
nuclear pellet was resuspended in 0.5 ml buffer containing 20
mM HEPES (pH 7.9), 20% glycerol, 0.6 M NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 2
mM DTT, and 0.5 mM phenylmethanesulfonyl
fluoride (PMSF) and was homogenized with 40 strokes. The homogenate was
incubated in an ice bath for 30 min with resuspension every 5 min and
then centrifuged at 15,000 rpm at 4 C in a JA 20 rotor for 30
min. The supernatant was collected and dialyzed against 100 vol of a
buffer containing 20 mM HEPES (pH 7.5), 20% glycerol, 100
mM NaCl, 0.2 mM EDTA, 5 mM
MgCl2, 2 mM DTT, 0.5 mM PMSF, and 1
nM Mib for 4 h with one change of buffer. The dialyzed
nuclear extract was centrifuged at the same speed for 20 min to remove
insoluble matter and was stored frozen at -100 C in small aliquots.
The protein concentration of nuclear extracts was measured using a
Bradford protein assay kit (Bio-Rad Laboratories, Inc.,
Hercules, CA).
Gel band shift experiments
Double-stranded oligonucleotides, containing the sequence of an
androgen-responsive element (ARE) in either the PSA or the hK2
promoters (18, 20) with XbaI 5' protruding ends, were
labeled with [
-32P] deoxycycidine triphosphate (3,000
Ci/mmol; Amersham Corp., Arlington Heights, IL), by Klenow
enzyme, to a specific activity of 8 x
107–108 cpm/µg. In vitro DNA
binding was determined by incubating 15 µg protein of nuclear
extracts in a buffer containing 20 mM HEPES (pH 7.9), 100
mM NaCl, 5 mM MgCl2, 0.1
mM EDTA, 12% glycerol, 4 mM DTT, and 1 µg
poly dI:dC, with or without unlabeled ds-oligo, in a 100-fold molar
excess for 30 min on ice before adding 20–30 fmol of a labeled
ds-oligo probe for an additional 10 min of incubation. For the
supershift assay, 1 mg of specific mouse anti-AR antibody
(PharMingen, San Diego, CA) was incubated with the LNCaP
nuclear extract, 30 min before in vitro DNA binding.
Nonimmune mouse IgG was included as a control. The above reaction
mixtures were electrophoresed in a prerun 5% polyacrylamide (29:1 of
acrylamide:bisacrylamide), 0.5 x TBE at 250 V for 1–1.5 h. Gels
were dried and autoradiographed.
Western blot analysis
For immunoblotting of the AR, LNCaP cells were grown under the
same conditions described above and were treated without or with
10-7–2x10-9 M T3 in
the presence or absence of Mib (10-9 M) plus
5% charcoal-stripped FBS. Nuclear extracts were prepared as described
above. Whole-cell lysates were prepared using a lysis buffer containing
62.5 mM Tris, 10% SDS, and 25 mM DTT. Protein
contents were quantified by either the Bradford assay (Bio-Rad Laboratories, Inc.) or the DC assay (Bio-Rad Laboratories, Inc.). Proteins were separated in 8% polyacrylamide SDS gels
and electrotransferred onto sheets of nitrocellulose (Bio-Rad Laboratories, Inc.). High-molecular-weight protein markers
(Amersham Corp.) were also included in gel
electrophoresis. The blots were blocked overnight with 5% nonfat milk
in TBST buffer (20 mM Tris-HCl, 137 mM NaCl,
and 0.1% Tween 20) and incubated with an AR antibody
(PharMingen) in 1:2000 dilution, and the same membrane was
probed separately with ß-tubulin (Sigma Chemical Co.).
The AR and ß-tubulin proteins were visualized by an antimouse IgG
antibody conjugated with horseradish peroxidase (1:2000 dilution),
enhanced chemiluminescence substrate (ECL, Amersham Corp.), and exposure to x-ray film. A densitometer was used to
quantify the specific bands of AR and tubulin. The AR level is
normalized by AR/tubulin.
Statistics
Statistical analysis was performed by ANOVA followed by
Student’s t test. A value of P < 0.05 was
considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, cells treated with T3
alone showed pronounced growth enhancement in a dose-dependent fashion
(P < 0.01). Interestingly, Mib seemed to attenuate the
growth enhancement achieved by T3 at 10-8 and
10-9 M. In the presence of Mib, significant
cell proliferative activity (P < 0.05) was achieved
only with high concentrations of T3 (10-7
M). We also examined whether T3 had a
stimulatory effect on the growth of an androgen-independent cell line,
PC-3. As shown in Fig. 1B, the growth of PC-3 cells was significantly
stimulated by T3, though high concentrations of
T3 were required (10-7–10-5
M). Because PC-3 cells lack androgen responsiveness,
experiments for the interactive effects of T3 and androgens
were not performed in this cell line.
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, T3, together with
androgen, enhanced the androgen-induced up-regulation of PSA expression
in a dose-dependent manner. In contrast, in the absence of androgen,
PSA expression was very low; and T3 alone had only a
marginal effect on its expression [2-fold at the highest concentration
treated (10-7 M)]. When hK2 expression was
examined (Fig. 2B), it was found that T3 had no additional
stimulation effect over that afforded with androgen alone. Moreover, in
the absence of androgen, T3 slightly inhibited hK2
expression. These data suggest that the effects of T3 on
the differentiation properties (i.e. PSA and hK2 expression)
may be different from the effects on the proliferation properties of
prostate cells.
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, Mib was a potent inducer of both PSA
and hK2 transcription activities, which is in agreement with previous
studies (20, 21). The PSA enhancer/promoter was induced by Mib to a
greater extent than that of hK2, which is in agreement with the above
observations on protein expression. T3 potentiated the
androgenic induction of the PSA enhancer/promoter (P <
0.05) but not that of hK2 (P = 0.65). T3
alone had no significant effect on transcriptional activity of either
the PSA or hK2 genes.
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). These results suggest that the
nuclear AR protein level is not increased by T3 during a
time when the transcription rate for the PSA gene is increased.
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, total AR protein was readily
increased by T3, at 2 x 10-9
M. Although androgens increased AR levels, as expected,
T3 (at all concentrations tested) did not further enhance
AR protein levels. Next, AR levels were measured in whole-cell lysates
and nuclear extracts from LNCaP cells, after treatment with
T3 at 10-7 M and/or Mib, in two
independent experiments (a representative result is shown in Fig. 5b).
When nuclear extracts were examined, it was found that the
immunostained band from the T3-treated cells migrated
faster than that from cells treated with either T3 and Mib
or Mib alone. Furthermore, T3 treatment resulted in a small
increase in AR expression in nuclear extracts, but no further
enhancement of AR levels was observed in the presence of androgens.
This is consistent with the results of the above band-shift assays.
Moreover, in nuclear extracts, androgen treatment resulted in a slower
migrating band on the gel. Because hyperphosphorylation of the AR,
induced by androgens, results in slower migration in SDS-PAGE gel (22, 23), these results are consistent with a change in the phosphorylation
status of the protein. T3 treatment did not seem to alter
the migration status of the AR in either whole-cell lysates or nuclear
extracts. Thus, it may not affect the phosphorylation status of the
protein.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the mechanism(s) by which thyroid hormones and androgens mediate the positive growth responses in prostate cells are still poorly understood, our present study suggests that the growth responses of LNCaP cells stimulated by thyroid hormone are not identical to that stimulated by androgens. Moreover, T3 can enhance androgenic induction of PSA expression, suggesting that T3 may further enhance the androgen-stimulated differentiation of prostate cells. Thus, the growth pathways regulated by the two agents may be, at least in part, separate. Moreover, T3 seems less potent for growth stimulation of PC-3 cells. Whether this is an indication that highly hormone-refractory cells become less thyroid hormone dependent is not clear.
We (20) and others (21, 27) have established that the expression of PSA and hK2 genes are regulated primarily at the transcriptional level by ligand-activated AR via ARE in the promoters of these two genes. As shown in our previous studies (20), the increase of mRNA of both PSA and hK2 parallel the increase of these two proteins after androgenic induction. Our present studies show that androgen-induced PSA protein expression was enhanced by T3, but under the same conditions, hK2 protein levels were not effected. Gene transfer experiments with PSA and hK2 enhancer/promoters showed consistent results with the protein expression of these two genes, suggesting that the T3 effects occur, at least in part, at the transcriptional level. Moreover, although T3 alone increased the total cellular AR level and slightly increased the nuclear AR level, it did not further increase the nuclear AR level over androgen treatment alone.
It is well established that androgens can enhance AR protein levels by increasing the half-life, as well as by stimulating the phosphorylation, of the AR (28, 29, 30). Whether the enhanced phosphorylation increases the stability of the AR is not clear. However, agonist-bound AR is more resistant to in vitro proteolytic digestion, indicating a conformational change of the AR induced by androgens. Thus, a conformation change may expose sites for phosphorylation by protein kinases (22). It has been suggested that phosphorylation may play a role in many diverse processes for nuclear receptors: ligand binding, translocation to the nucleus, dimerization, DNA binding, and interaction with other proteins (22). Phosphorylation of the AR by androgens results in an altered migration on an SDS-PAGE gel, as evidenced in previous reports (23) and suggested by this study. T3 alone did not seem to change the phosphorylation status of the AR. This may explain why T3 alone did not exhibit significant effects on transactivation of either the PSA or hK2 genes. Furthermore, although T3 alone increased AR protein expression, the expression of androgen-regulated genes, such as PSA and hK2, was not drastically increased by T3. This further suggests the importance of androgen-induced phosphorylation. Moreover, how T3 alone can affect AR levels remains to be elucidated.
Because T3 did not seem to stimulate either phosphorylation of the AR or activation of transcription of the PSA and hK2 genes, it is puzzling why T3, in the absence of androgens, could enhance the expression of PSA, yet repress the expression of hK2. Whether T3 can modulate the secretion of PSA and hK2 proteins remains to be determined. Nonetheless, the effects of T3 on androgenic induction of these two genes at the transcriptional levels cannot simply be explained by the levels of AR protein. The question, therefore, arises as to whether T3 enhances PSA transcription via the T3 receptor and its cognate responsive elements in the PSA gene. If this is the case, perhaps AR binding to the PSA promoter is a prerequisite for T3 receptor-mediated enhancement of transactivation. Currently, we are vigorously pursuing a study for identifying the T3 receptor binding site in the PSA promoter.
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Acknowledgments |
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Footnotes |
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2 Antibodies to hK2 are patented; and G. G. Klee, D. J.
Tindall, and C. Y. F. Young receive royalties from these
patents.
Received July 30, 1998.
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