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The Prostate 28: 182- I 94 ( 1996)
Control of LNCaP Proliferation and Differentiation:
Actions and Interactions of Androgens,
I a,25-Dihydroxycholecalciferol, All-Truns Retinoic
Acid, 9-Cis Retinoic Acid, and Phenylacetate
Murielle Esquenet, Johannes V. Swinnen, Walter Heyns, and
Guido Verhoeven
laboratory for Experimental Medicine and Endocrinology, Department of Developmental
Biology, Onderwijs en Navorsing, Gasthuisberg, Catholic University of Leuven,
leuven, Belgium
ABSTRACT:
There is increasing evidence that growth and differentiation of prostatic
carcinoma cells may be modulated not only by androgens and growth factors but also by
vitamin D, retinoids, and phenylacetate (PA). The latter agonists may have a role in the
prevention and therapy of prostate cancer but their exact therapeutic potential remains
unclear. Since both retinoids and vitamin D act via nuclear receptors, the same way androgens do, we studied the interactions of these compounds with androgen-induced proliferation and differentiation using LNCaP cells as a model of androgen-responsive tumor cells.
PA was included because of its suspected different mode of action. [,H]-thyrnidine incorporation was used as a measure of proliferative activity, secretion of prostate-specific antigen (PSA) as a measure of differentiated function.
The present data show that la,25-dihydroxycholecalciferol(VD,), all-trans retinoic acid
(atRA), 9-cis retinoic acid (~cRA),and PA stimulated LNCaP cell-differentiated function in
the presence or absence of androgens. The effects on cell growth were more complicated. In
the absence of androgens growth stimulatory effects were observed for the retinoids and
under some conditions for VD,. These effects were limited, however, and tended to be more
pronounced at low cell densities. In the presence of androgens nearly exclusively growth
inhibitory effects were observed. On a molar basis VD, was the most effective antiproliferative agonist (ED,, =
M). It completely neutralized the stimulatory effects of androgens. Growth inhibition was not due to a decrease in the concentration of androgen receptor: whereas atRA, 9cRA, and PA did not alter androgen receptor levels, VD, provoked a
twofold increase. Neither in the presence nor in the absence of androgens did we observe
any cooperativity in the growth stimulatory or inhibitory effects of VD,, atRA, or 9cRA. To
test whether treatment with any of the studied agonists resulted in a phenotypic reversion
and sustained growth arrest, LNCaP cells were pretreated with VD,, atRA, 9cRA, or PA for
6-12 days and reseeded at equal densities as untreated cells. In all cases tested [,H]-thyrnidine incorporation was restored within 6 days suggesting that none of these compounds
caused irreversible growth inhibition. 0 1996 Wiley-Liss, Inc.
KEY WORDS:
prostate cancer, vitamin D, retinoids, differentiation therapy, prostatespecific antigen
INTRODUCTION
Prostate cancer is becoming an increasingly important problem in our aging society [l].The mechanisms
the Origin and the
Of the
disease remain poorly understood. It has been well
0 1996 Wiley-Liss, Inc.
Received for publication August 9, 1994; accepted December 6,
1994.
Address reprint requests to G. Verhoeven, Laboratory for Experimental Medicine and Endocrinology, Department of Developmental Biology, Onderwijs en Navorsing, Gasthuisberg, Catholic University of Leuven, B-3000 Leuven, Belgium.
Growth and Differentiation of LNCaP Cells
established, however, that during the initial stages of
tumor development androgens play at least a permissive role. For many years androgen ablation or blockade has become the therapy of choice for the treatment of metastatic carcinoma [2]. Unfortunately,
tumors tend to become androgen insensitive and at
this point endocrine therapy fails.
In search for new therapies it has become increasingly clear that apart from androgens several other
steroidal and nonsteroidal factors such as vitamin D,
retinoids, and phenylacetate (PA) influence proliferation and differentiation of prostatic carcinoma cells
studied in vitro [3-lo]. Epidemiologic investigations
have presented evidence that vitamin D as well as
vitamin A may protect against clinical prostate cancer
[ll-131 and it has been suggested that both compounds might offer new avenues for the treatment of
androgen-dependent as well as androgen-independent prostate cancer.
A number of tumor cell lines have proven useful in
the study of the response of prostatic carcinoma cells
to growth factors [14-191. LNCaP cells, however, are
the only ones that remain androgen responsive in
vitro [20-221. This cell line was established by
Horoszewicz et al. [23] from a lymph node metastasis
of a patient who developed refractory disease following androgen-deprivation therapy. LNCaP cells respond to androgens with increased proliferation and
some of their differentiated functions such as production of acid phosphatase (PAP) and prostate-specific
antigen (PSA) are also androgen regulated [24-271.
LNCaP cells have high concentrations of an androgen
receptor, but a point mutation in the ligand binding
domain results in an aberrant response to progestins,
estrogens, and antiandrogens [28-311.
Recently it has been demonstrated that LNCaP
cells have receptors for other members of the nuclear
receptor family such as vitamins D and A. Proliferative as well as antiproliferative effects of the corresponding ligands have been demonstrated and it has
been shown that both vitamins D and A are able to
stimulate differentiated functions of LNCaP cells
[3,4,71.
In the present paper we used the LNCaP model to
study whether there are any interactions between androgens and vitamin D or vitamin A, ligands for two
other members of the nuclear receptor family. Moreover, we explored whether 9-cis retinoic acid (~cRA),
which modulates the effects of vitamins D and A in a
number of other systems [32], influences the effects
of these agonists in LNCaP cells. Finally, since it has
been claimed that differentiation of prostate cancer
cells by agonists such as PA might irreversibly change
the malignant behavior of invasive prostate tumor
cells [lo], we investigated the reversibility of the ef-
183
fects of vitamin A, vitamin D, and PA on the proliferation of LNCaP cells.
MATERIALS AND METHODS
Cell Culture
The LNCaP human prostatic cancer cell line was
obtained from the American Type Culture Collection
(ATCC, Rockville, MD). The cells were routinely
maintained in RPMI-1640 medium supplemented
with 10% fetal calf serum (FCS), 3 mM L-glutamine,
100 pg/ml streptomycin, and 100 U/ml penicillin. All
media and supplements were obtained from GIBCO
BRL (Paisley, Scotland). The cultures were trypsinized
twice weekly and were maintained in a humidified
atmosphere of 5% C 0 2 in air. Where indicated FCS
was pretreated with dextran-coated charcoal (DCCFCS; 0.25% [w/v] dextran; 2.5% [wh] charcoal) [33].
[3H]-Mibolerone, unlabeled mibolerone, and
R1881 (methyltrienolone) were purchased from Dupont-New England Nuclear (Boston, MA). la,25dihydroxy-cholecalciferol (VD,) was obtained from
Solvay Duphar (Weesp, Holland). g-cis-retinoic acid
(9cRA) was kindly provided by Dr. Uskokovic (Hoffmann-La Roche, Nutley, NJ). All-trans retinoic acid
(atRA) and phenylacetate (PA) were obtained from
Sigma Chemical Co. (St. Louis, MO).
Cell Proliferation Assays
Cell proliferation was evaluated either by measurements of [,H]-thyrnidine incorporation, by cell counting, or by DNA measurements.
For the [3H]-thymidineincorporation,experiments,
cells were seeded in 96-well multidishes (Nunc,
Roskilde, Denmark) at a density of 5 x lo3 cells per
well in RPMI-1640 with 10% FCS. The next day the
medium was changed to RPMI-1640 supplemented
with 10% DCC-FCS. Fourty-eight hours later the medium was changed to RPMI-1640 with 5% DCC-FCS
supplemented with the compounds of interest. Steroids, vitamin D, and vitamin A derivatives were
added in ethanol. The final ethanol concentration did
not exceed 0.1%. PA was dissolved in distilled water,
brought to pH 7.0 by the addition of NaOH, and
stored in aliquots at -20°C. Depending on the experiment, incubation was continued for 3 up to 6 days.
For incubation periods of 6 days, the medium was
changed after 3 days. After this incubation, [3H]-thymidine (specific activity: 2 Ci/mmol; ICN, Irvine, CA)
was added (1 pWwe11). Six hours later, the media
were removed; the cultures were washed twice with
100 pl ice-cold phosphate buffered saline (PBS),
trypsinized, and harvested on Whatman filter paper
(grade 934AH, Whatman, Maidstone, England) using
184
Esauenet et al.
a multiple-cell harvester (Nunc). The filters were collected and counted in 3 ml counting scintillant (InstaGel, Packard Instrument Company, Meriden, CT).
In some experiments, proliferation was evaluated
by cell counting. To this end 6 x lo4 cells were seeded
in 12-well multidishes. The culture conditions were
identical to those described for the [3H]-thymidine
incorporation experiments. After the incubation periods, cells were detached by trypsinization and
counted using a Coulter Counter (model ZB.0067).
DNA content of the cultures was measured using
the method of Labarca and Paigen [34].
Study of the Reversibility of the Effects of VD,,
atRA, 9cRA, or PA on LNCaP Cell Proliferation
To study the reversibility of the growth-suppressing effects of VD,, RA, and PA, LNCaP cells were
seeded in 80 cm2 flasks at a density of 1.25 X lo6 cells
in RPMI-1640 with 10% FCS. After 24 hr the medium
was changed to RPMI-1640 with 5% FCS supplemented or not with the test compounds. The medium
was changed every 3 days. This pretreatment was
continued for 6 or 12 days. Thereafter, cells were
trypsinized, counted in a Biirker Chamber, and reseeded in 96-well plates at a density of 5x103 cells
per well. These cells were cultured for an additional
period of 3-6 days in RPMI-1640 supplemented with
5% FCS, 5% DCC-FCS, or 5% DCC-FCS + lo-'' M
R1881. After this incubation time, [,H]-thyrnidine incorporation was studied as described above.
Study of the Effects of VD,, RAs, and PA on
Androgen Receptor Binding
Androgen receptor binding was studied in intact
LNCaP cells pretreated with various test compounds
using a single point displaceable binding assay as described previously [35]. Briefly, cells were plated in
6-well plates in medium with 10% FCS and allowed to
adhere overnight. The medium was changed to medium containing 10% DCC-FCS and 48 hr later pretreatment with the different test compounds was
started in RPMI-1640 supplemented with 5% DCCFCS. After an incubation time of 6 days, cultures were
carefully washed with serum-free medium and incubation was continued for an additional 24 hr in serumfree medium in the presence of the respective test
compounds. Finally, after three more washes with
serum-free medium, androgen receptor binding was
measured. To this end [3H]-mibolerone was added at
a concentration of 5 x lop9 M, in the absence (tracer)
or presence (overload) of unlabeled mibolerone (lop6
M) and the cells were incubated for 24 hr at 37°C. At
the end of the incubation the medium was discarded
and the cells were washed three times with ice-cold
PBS containing 0.2% bovine serum albumin. Cells
were recovered in 1 ml of 1%(v/v) Triton X-100 in 0.1
N NaOH. After transfer to scintillation vials, 3 ml of
counting scintillant was added and the radioactivity
was counted. Displaceable binding was calculated and
expressed per milligram cell protein. Protein concentrations were measured using the Coomassie protein
assay reagent (Pierce, Rockford, IL).
Statistics
Determination of PSA Secretion by LNCaP Cells
The secretion of PSA was used as a parameter of
differentiated cell function in LNCaP cultures. To this
end LNCaP cells were seeded at a density of 6 x lo4
cells per well in 12-well multidishes in RPMI-1640 medium supplemented with 10% FCS. The following
day the medium was changed to RPMI-1640 containing 10% DCC-FCS. Fourty-eight hours later medium
with 5% DCC-FCS and supplemented with the indicated concentrations of the studied test compound
was added to each well. After 3 days, the media were
replaced by media containing the same test compounds but supplemented with lo-'' M R1881 as indicated. The cells were cultured for another 3 days,
the conditioned media were collected, centrifuged to
remove residual cells (800 rpm; 10 min), and stored at
-20°C. PSA levels were determined using the Tandem-R immunoradiometric assay (Hybritech, San Diego, CA) according to the manufacturer's instructions. To account for differences in cell number,
results were expressed per microgram DNA.
Statistical analysis was performed using analysis of
variance followed by Tukey's t test. A value of Pc0.05
was considered statistically significant.
RESULTS
Effects of Androgens on the Proliferation of
LNCaP Cells
In a first series of experiments optimal conditions
for the study of the effects of androgens on the proliferation of LNCaP cells were delineated. Cells were
cultured in DCC-FCS to prevent interference of serum-derived androgens. Increasing concentrations of
the synthetic androgen R1881 were added since natural androgens are rapidly metabolized in LNCaP
cells [20]. After various periods of time [3H]-thymidine incorporation as well as cell number were measured as a parameter of proliferative activity.
In agreement with previous reports [20-221 [,HIthymidine incorporation showed a biphasic response
to increasing concentrations of R1881 (Fig. 1A). Max-
Growth and Differentiation of LNCaP Cells
185
h
m
50
? IL-1
2:
0
C
-12-11
.
-10 - 9
-8 -7
-6
B
T
A
T
L
C
-12-11
-10 - 9
-8
-7
-6
R 1 8 8 1 ( l o g M)
Fig. 1.
Effect of androgen concentration on the proliferation of
LNCaP cells. LNCaP cells were seeded in 96-well plates at a density of 5 x I O3 cells per well for [3H]-thymidineincorporation (A)
or at a comparable density of 6 X lo4 cells per well in a 12-well
plate for cell counts (B). [3H]-thymidine incorporation (A) was
measured after 24 hr (O),48 hr (V), 72 hr (W), and 96 hr (A)of
incubation in the presence of increasing concentrations of the synthetic androgen R I 88 I. Cell counts (B) were determined after 72
hr (0)
and 6 days (0)of incubation with R1881. Results shown
represent the mean k SD of incubations performed in triplicate.
imal stimulatory effects were observed at a concentration of lo-'' M. At higher concentrations [,H]-thymidine incorporation dropped sharply. No changes
in optimal R1881 concentrations were observed as a
function of time of incubation, but the effect was
more pronounced after longer incubation periods
(Fig. 1A). It may be noted that a dose-dependent increase in [,H]-thymidine incorporation was already
apparent after an incubation period of 24 hr. Maximal
incorporation (four to fivefold stimulation as compared to the control) was observed after 72 hr of incubation. A similar dose-response relationship was
observed when cell numbers were used as a parameter of the proliferative activity of LNCaP cells (Fig.
1B). However, more prolonged exposure to androgens (6 days) was required to document this response. For these reasons [,H]-thymidine incorporation after 3 days of incubation was selected to
evaluate androgen effects in all subsequent experiments.
and the factors determining the nature of these effects are only partially understood.
To analyze the actions of the mentioned compounds in detail LNCaP cells were seeded at three
different densities (lo3, 5 x lo3, and lo4 cells per well)
and exposed for six days to increasing concentrations
of VD,, atRA, 9cRA, and PA. All experiments were
performed in DCC-FCS. Moreover, eventual interactions of the test compounds with androgens were
evaluated by adding a maximal stimulatory concentration of R1881 (lo-'" M) to half of the culture wells
during the last 3 days of incubation. [,H]-thymidine
incorporation was used as a measure of proliferative
activity.
The results obtained with cells seeded at a concentration of 5 x lo3 cells per well are summarized in Figure 2. Under these conditions VD, inhibited [,H]-thymidine incorporation. This effect became evident
from a concentration of lop9 M on. In the absence of
androgens [,H]-thymidine incorporation was reduced to approximately 50% of the control value. In
the presence of androgens a reduction to less than
30% of the control value was observed. At concentrations of VD, equal or higher than lop8 M the stimulatory effect of androgens was completely neutralized. Roughly the same effects were observed when
LNCaP cells were seeded at higher densities (lo4
cells/well). At lower cell densities (lo3cells/well)stimulatory effects of VD, were noted at a concentration
Effects of VD,, RAs, and PA on C3H+Thymidine
Incorporation in LNCaP Cells Grown in the
Presence or Absence of Androgens
As summarized in the introduction, evidence has
been presented that vitamin D, vitamin A, and PA
may modulate growth and differentiation of prostate
tumor cells and cell lines [3-lo]. The effects of the
first two compounds may be stimulatory or inhibitory
186
Esquenet et al.
40
//'
30-
"D3
I
".
1
i-
atRA
i'
k
\
20 -
lo
I
-12
L
C
-10
-8
-6
-
30
-
20
- 10
n-
4
C
\.
- 40
-12
-10
-8
-6
++
J
75
60
45
\*
5
C
-12
-10
-8
-6
C
-5
-4
-3
-2
CONCENTRATION ( l o g M )
Fig. 2. Dose-dependent effects of VD,, atRA, 9cRA. and PA on
[ 'HI-thymidine incorporation by LNCaP cells in the presence o r
absence of androgens. Cells were seeded in 96-well plates at a
density of 5 x lo3 cells per well. The cultures were treated as
described in Materials and Methods. [ 'HI-thymidine incorporation
was measured after 6 days of incubation with increasing concen-
trations of the indicated compounds in the absence of androgens
(0)or in the presence of 10- l o M R I88 I (0)during the last 3
days of culture. Results shown represent the mean SD of incubations performed in triplicate. Values which are statistically different (W0.05)from the respective control values are indicated by
an asterisk.
of lo-" M. These effects were limited (25% in the
presence of androgens and 100% in the absence of
androgens) and were replaced by inhibitory effects at
higher concentrations of VD, (not shown).
In the absence of androgens atRA stimulated [,HIthymidine incorporation at concentrations ranging
from lo-'' up to
M. In the presence of androgens limited stimulatory effects were observed at
M and lo-'' M whereas inhibitory effects became obvious at concentrations exceeding lop8 M.
Again stimulatory effects tended to be more pronounced when cells were seeded at lower densities.
In the presence of androgens, however, inhibitory
effects were consistently observed at concentrations
equal or higher than lop8 M (not shown).
9cRA slightly stimulated [,H]-thymidine incorporation in the absence of androgens at concentrations
between lo-'' and
M. Against this effect be-
came more pronounced at lower cell densities (lo3
cells/well; not shown). At higher concentrations of
9cRA inhibitory effects prevailed particularly in the
presence of androgens.
PA provoked a dose-dependent inhibition of [,HIthymidine incorporation at all cell densities studied
both in the presence and absence of androgens. In
the presence of androgens the effect became apparent
at a concentration of lop5 M. In the absence of androgens a concentration of
M was required.
To explore whether VD,, atRA, and 9cRA might
display any cooperativity in their effects on [,H]-thymidine incorporation, LNCaP cells were exposed to
lop9 M VD,,
M atRA, and
M 9cRA or to
combinations of these agonists. The effects were
studied both in the presence and absence of androgens. As illustrated in Table I no cooperativity was
observed neither for the stimulatory effects observed
*
187
Growth and Differentiation of LNCaP Cells
TABLE 1. Effect of Combined Compounds on Basal and Androgen-Induced
Proliferation of LNCaP Cellst
Control
lop9 M VD,
lo-' M atRA
10-7 M ~ C R A
l o p 9 M VD, + lop8 M atRA
10-9 M VD, + 10-7 M ~ C R A
lo-' M atRA +
M 9cRA
No androgen
lop'' M R1881
100 f 1
87 ? 6
168 ? 10'
171 ? 9*
118 ? 5*
153 t 4*
159 ? 10*
100 f 8
44 ? 1*
37 t 4*
30 ? 1'
30 ? 2*
25 ? 5*
28 -C 3*
~~
~
'LNCaP cells were seeded in 96-well plates at a density of 5 x lo3 cells per well and cultured
as described in Materials and Methods. [3H]-thymidine incorporation was measured after 6
days of incubation with the mentioned compounds. Where indicated lo-''' M R1881 was
added during the last 3 days. Results shown represent the mean 2 SD of incubations performed in triplicate and are expressed in % compared to the respective controls.
Values which are statistically different ( R 0 . 0 5 ) from the respective control values.
in the absence of androgens nor for the inhibitory
effects observed in the presence of R1881.
h
m
I
0
4
Influence of VD,, atRA, 9cRA, and PA on the
Responsiveness of LNCaP Cells to Androgens
In order to explore whether VD,, RAs, or PA might
affect the characteristic biphasic dose-response curve
of [,H]-thymidine incorporation to androgens, LNCaP
cells were exposed to increasing concentrations of
R1881 in the presence or absence of lop8 M VD,,
M atRA, lop6M 9cRA, and 5.10p3M PA. As shown in
Figure 3 basal and androgen-stimulated [,H]-thyrnidine incorporation was suppressed but maximal androgen responses were still observed at lo-" M
R1881. The decrease in androgen responsiveness was
apparently not related to a change in androgen receptor concentration. In fact, as summarized in Table
11, VD, provoked a 2.5-fold increase rather than a
decrease in androgen receptor levels whereas the
other compounds tested displayed only marginal effects.
Effects of VD,, atRA, 9cRA, and PA
on PSA Secretion
PSA was monitored as a parameter of the differentiated secretory functions of LNCaP cells. As expected androgens markedly stimulated PSA secretion
(Table 111). In the absence of androgens all concentrations of VD,, atRA, 9cRA, and PA tested displayed
stimulatory effects. A twofold increase was observed
with VD, and PA whereas a fourfold increase was
noted with the RAs. Even in the presence of R1881 a
50% increase in PSA secretion was observed with all
compounds tested.
25
K
x
E
za
20
15 -
10 -
5 -
I
z
I
C
-12
-11
I
-10
% -
-9
-8
-7
R1881 ( l o g M )
.,
Fig. 3. Dose-response curves of R I 881 in the presence of inhibitory concentrations of VD,, atRA, ScRA, and PA. 5 x IO3 LNCaP
cells were seeded in 96-well plates and incubated as described in
Materials and Methods. After a 3-day incubation period with each
of the test compounds (0,
control; V, lo-* M VD,;
lo-' M
atRA; 0,
M 9cRA; A, 5.10-3 M PA), the medium was
changed t o medium containing both the test compound and increasing concentrations of R I88 I. [ ,H]-thymidine incorporation
was measured 3 days later. Values represent the mean ? SD of
incubations performed in triplicate.
Reversibility of the Growth Inhibitory Effects of
VD,, atRA, 9cRA, and PA
In order to explore whether the investigated compounds might induce terminal differentiation and ir-
188
Esquenet et al.
TABLE II. Effect of VD, atRA, 9cRA, and PA on
Androgen Receptor Level in LNCaP Cellst
Receptor binding
(% of control)
Control
VD,
lo-' M
lo-* M
M
100 ? 7
244
243
264
* 38
?
2
6
43
atRA
lo-* M
137 5 9
120 1
*
143 * 22
M
9cRA
lop7 M
lo-' M
106 ? 7
PA
*
10-3 M
5.1OP3 M
110 6
84 ? 12
+LNCaPcells were seeded in 6-well plates and incubated as described in Materials and Methods. After an incubation period of 6
days in the presence of the mentioned compounds androgen receptor binding was measured by a single point monolayer binding
assay. Results shown are expressed as percent compared to control
and are expressed as mean SD. Receptor binding in the control
was 617 2 53 fmol/mg protein.
*
reversible growth arrest of prostatic tumor cells, LNCaP cells were exposed for 6 days to VD,, atRA,
9cRA, and PA. All pretreatments were performed in
5% FCS. Thereafter control cultures and pretreated
cells were trypsinized and identical numbers of viable
cells were seeded in multidishes. The growth potential of these cells was examined by exposing them to
DCC-FCS in the absence or presence of R1881 or to
FCS. [3H]-thymidine incorporation or cell DNA was
measured at the end of a 3-day or 6-day incubation
period.
Pretreatment with VD, reduced [,H]-thymidine incorporation at the end of the 3-day incubation period
(Fig. 4). A limited stimulatory effect of R1881 and FCS
could still be observed but this effect was not reflected in an increase in the DNA content of the cell
cultures (Table IV). At the end of the 6-day incubation
period [,H]-thymidine incorporation was further decreased in cultures maintained in DCC-FCS. A
marked response to R1881 and FCS was observed,
however, and in pretreated cells grown in FCS [3H]thymidine incorporation equalled that observed in
control cultures. The DNA contents of the cultures
also reflected a marked response to R1881 and FCS
although the levels of DNA remained clearly below
those observed in nonpretreated cells. Similar results
were observed for cells pretreated with PA. In this
case [3H]-thymidine incorporation after 6 days was
TABLE 111. Effect of VD,, atRA, 9cRA. and PA on PSA
Secretion by LNCaP Cellst
No androgen
Control
VD3
M
lo-* M
M
0.216
* 0.044
lo-'' M R1881
2.103
?
5
*
0.016'
0.074'
0.019'
3.759 ? 0.509'
3.556 5 0.011'
2.860 -+ 0.149'
0.876
0.882
0.874
2
0.040'
3.578 0.051'
3.115 t 0.297'
3.052 5 0.413'
* 0.028'
* 0.073'
0.927 5 0.123'
1.076 ? 0.14T
0.881 ? 0.080'
PA
10-3 M
5.10-3 M
0.291
0.478
0.403
0.324
atRA
lop9 M
lo-* M
lop7 M
9cRA
lop8 M
10-7 M
M
2
0.440
0.778
* 0.070'
?
0.049'
*
3.314
3.281
3.448
3.099
3.456
5
?
*
0.267'
0.443'
0.725'
0.432'
* 0.150'
+LNCaP cells were incubated in the presence of varying concentrations of the indicated compounds for 6 days as outlined in Materials and Methods. Conditioned media of the last 3 days were
collected and PSA levels were determined. DNA content of the
cultures was determined simultaneously. Values are expressed in
ng/pg DNA. Results shown are expressed as the mean k SD of
incubations performed in triplicate.
"Values which are statistically different (P<0.05) from the respective control.
not significantly different in control cultures and pretreated cultures and the amount of DNA in the latter
cultures was close to that observed in the control cultures.
It may be noted that pretreatment with atRA
M) in medium supplemented with FCS resulted in an
inhibition of [,H]-thymidine incorporation (Fig. 5,
3-day incubation period) rather than in a stimulation
as observed in medium supplemented with DCC-FCS
(Fig. 2). After 6 days of incubation the inhibition disappeared. At lop7 M the inhibitory effects of atRA
became even more obvious and at that concentration
a 50% reduction in cell DNA content was observed at
the end of the 6-day incubation period (Table IV). A
comparable degree of growth inhibition was observed with l o p 6 M 9cRA.
To investigate whether a more prolonged pretreatment might produce irreversible growth inhibition,
LNCaP cells were exposed for 12 days to
M VD,.
This resulted in a more marked inhibition of [3H]thymidine incorporation at the end of the 3-day incubation period (Fig. 6). Moreover, at this time point
cells did not respond to androgens or FCS. After 6
days of incubation, however, only cells maintained in
DCC-FCS still displayed diminished [3H]-thymidine
incorporation, whereas in LNCaP cells treated with
Growth and Differentiation of LNCaP Cells
189
Pretreatment with VD, ( 6 days)
60
t
T
a
P r e t r e a t m e n t with PA ( 6 days)
G
0
I inc. 6
inc. 3 d
2
E
La
inc. 6 d
inc. 3 d
d
T
45
T
D
15
L
DCC-FCS
DCC-FCS
+
Rl88l
FCS
DCC-FCS
DCC-FCS
+
FCS
Rl88l
Fig. 4. Effect of retrea ment with VD, or PA on LNCaP cell
proliferation in the presence and absence of androgens. LNCaP cells
were cultured for a period of 6 days in the absence (open bars) or
in the presence of of lop8 M VD, or 5.10-' M PA (hatched bars)
as described in Materials and Methods. Thereafter the cells were
reseeded in 96-well plates at a density of 5 x lo3 cells per well.
icubation (inc.) was continued for 3 or 6 days in RPMI-I 640 supplemented with 5% DCC-FCS, 5% DCC-FCS
10- M R I88 I,
or 5% complete FCS. At the end of this period (3H]-thymidine
incorporation was measured. Results shown represent the mean ?
SD (n = 3) for a representative experiment.
R1881 or FCS [,H]-thyrnidine incorporation was completely restored.
[3H]-thymidine incorporation at a concentration of
lo-'' M. A 3-day incubation period with lo-'' M
R1881 was selected for further experiments. The remarkable shape of this dose-response curve remains
poorly understood. The present data clearly show
that the shape of this curve was not affected by the
duration of androgen exposure (24-96 hr). Moreover,
it is worth noting that a classical hyperbolic response
curve was observed for androgen-stimulated parameters of differentiated function of LNCaP cells such as
the induction of PSA and diazepam-binding inhibitor
(DBI) mRNA [39].
In the absence of androgens the studied agonists
(VD,, atRA, 9cRA, PA) displayed variable effects on
cell proliferation. PA consistently inhibited [,H]-thymidine incorporation with an ED,, value of 5 mM
comparable to that reported by Samid et al. [lo]. The
effects of vitamin D and retinoids were more variable
and depended not only on the concentration of these
agonists but also on cell density which may explain
some of the controversy in the literature [3,4,7]. VD,
DISCUSSION
One of the main aims of this study was to explore
the interactions between androgens, the main
growth-promoting hormones for prostate tumor
cells, and VD,, atRA, 9cRA, and PA, a series of agonists with potential antiproliferative activity. LNCaP
cells were selected for these experiments since they
represent the only widely employed system which
allows the study of androgen-regulated growth of
prostate cancer cells in vitro [20-231.
The proliferative response of LNCaP cells to androgens markedly depends on culture conditions
[36-381 and accordingly, optimal conditions to quantitate this response were first delineated. In agreement with previous reports [20-223 cells maintained
in DCC-FCS displayed a steep biphasic dose-response curve after 3 days of incubation with the synthetic androgen R1881 with a maximal stimulation of
+
'
190
Esquenet et al.
TABLE IV. Influence of 6-Day Pretreatment With Various Agonists on Basal and Androgen-Induced Cell Growth*
3-Day incubation
Pretreatment
Control
VD3
lo-' M
10-7 M
atRA
lo-' M
M
9cRA
M
lo-' M
PA
M
5.10-" M
DCC-FCS
DCC-FCS
6-Day incubation
+ R1881
FCS
* 0.22
3.44 t 0.37
4.50 t 0.30
1.62 ? 0.08
2.11 f 0.17
1.76 0.32
1.88 f 0.09
1.88 f 0.22
1.56 ? 0.04
1.91 f 0.21
1.93 0.13
*
2.19 t 0.19
1.99 0.09
1.64 f 0.21
n.d.
2.66 f 0.17
1.88 0.09
3.75
2.08
*
5.51
DCC-FCS
5.41
1.02
DCC-FCS
+ R1881
* 2.53
2.95 * 0.66
FCS
8.68
14.7 f 2.79
1.44 f 0.33
2.04 f 0.47
4.46 t 0.37
5.11 t 0.44
6.69 f 0.17
2.77 0.65
1.96 f 0.15
1.44 f 0.35
1.89 t 0.14
5.48
4.23
2.37 f 0.25
n.d.
2.66 t 0.27
n.d.
2.76
1.52
* 0.74
* 0.29
0.13
3.92 t 0.18
1.99 f 0.95
2.76 f 0.27
3.00 t 0.46
*
f
* 0.25
*
f
* 0.97
f
0.48
8.24
4.44
f
f
0.22
0.50
8.66
6.76
f
0.59
* 0.31
10.88 ? 0.57
7.62 t 0.32
* 1.59
* 0.62
10.07 * 0.49
11.28
5.78
10.58 ? 0.06
*LNCaP cells were pretreated with the mentioned test compound for a period of 6 days as outlined in Materials and Methods. Cells were
reseeded in 12-well multidishes at a density of 6 x lo4 cells per well and were incubated in RPMI-1640 medium supplemented with 5%
DCC-FCS, 5% DCC-FCS + lo-'' M R1881, or full serum. Three or 6 days later DNA content of the cultures was determined. Values are
expressed in kg DNA/well. Results shown represent the mean ? SD of incubations performed in triplicate. n.d., not determined.
displayed mainly growth inhibitory effects. At low
cell densities, however, limited stimulatory effects
(up to twofold) were observed. These effects were
maximal at lo-" M. Stimulatory effects were observed with both atRA and 9cRA over a broad range
of concentrations (lo-'' up to lop6 M and lo-" M up
to
M, respectively). Again these effects tended
to be more pronounced at lower cell densities and to
decrease at higher cell densities or at higher concentrations of the retinoids. In general it should be
stressed that in comparison with androgens the observed proliferative effects of vitamin D and retinoids
were variable and limited.
In the presence of androgens growth-stimulating
effects became even less prominent (atRA) or disappeared (9cRA). Interestingly, concentrations of atRA
with obvious stimulatory effects in the absence of androgens (e.g., lop7 M) caused 50% growth inhibition
in the presence of these hormones, whereas limited
stimulatory effects became evident at very low concentrations of atRA which were inactive in the absence of androgens (Fig. 2). A comparable complex
response pattern to RA was observed by Fong et al.
[7] using dihydrotestosterone (DHT) as an androgen.
The antiproliferative effects of vitamin D and retinoids were clearly dose dependent. On a molar basis
VD, was by far the most efficient growth inhibitor
investigated (ED,, approximately lop9 M). This compound completely neutralized the proliferative effect
of androgens. Some more variability was observed
for atRA and 9cRA (ED,, between lo-' M and lop7 M
and between lop7 M and
M, respectively). The
ED,, for PA was approximately lop3 M. It may be
concluded that the antiproliferative activities of the
studied compounds are maintained and become even
more pronounced in the presence of androgens. The
mechanism of this growth inhibition remains elusive.
The present data do not support any direct interaction with the androgen-response apparatus of the
cells. Androgen receptor levels were maintained in
the presence of atRA, 9cRA, and PA and were even
markedly increased in the presence of VD,. Moreover, in the presence of suboptimal concentrations of
the inhibitors the remarkable shape of the dose-response curve to androgens remained unchanged:
maximal [3H]-thymidine incorporation was still observed at lo-'' M R1881. Finally, the studied compounds opposed the growth-promoting effects of androgens on LNCaP cells but enhanced their effects on
differentiated secretory functions.
In fact, in contrast with the complexity of their
effects on the proliferation of LNCaP cells, VD,,
atRA, 9cRA, and PA stimulated PSA secretion under
all the conditions investigated. Stimulatory effects on
PSA secretion have previously been reported for VD,
and atRA [4,7]. Our data show that these effects were
modest when compared with those of androgens.
They remained significant, however, even in the
presence of lo-'' M R1881. Moreover, for atRA and
9cRA, they could be observed both under conditions
Growth and Differentiation of LNCaP Cells
191
P r e t r e a t m e n t with atRA ( 6 d a y s )
I
inc. 3 d
60
h
0
4
451 k
':
E
a
U
a
30
e:
P r e t r e a t m e n t with 9 c R A ( 6 d a y s )
U
Pnc.
0
5
w
5
6o
E
E
45
inc. 6 d
t
DCC-FCS
DCC-FCS
+
FCS
DCC-FCS
R1881
DCC-FCS
+
FCS
Rl88l
Fig. 5. Effect of pretreatment with atRA or 9cRA on LNCaP cell
proliferation in the presence or absence of androgens. LNCaP cells
were cultured for a period of 6 days in the absence (open bars) or
in the presence of lo-* M atRA or
M 9cRA (hatched bars).
Thereafter the cells were reseeded in 96-well plates at a density of
5 X lo3 cells per well. Incubation (inc.) was continued for 3 or 6
days in RPMI- I640 supplemented with 5% DCC-FCS, 5% DCCFCS
lo-'' M R1881, or 5% complete FCS. A t the end of this
period [ 'HI-thymidine incorporation was measured. Results shown
represent the mean ? SD (n = 3) for a representative experiment.
which stimulate proliferation and under conditions
which inhibit proliferation indicating that proliferation and differentiation are not mutually exclusive.
Little is known on the mechanisms by which androgens, vitamin D, and retinoids influence the proliferation of LNCaP cells. All these factors are ligands
for members of a superfamily of nuclear receptors
that modulate gene expression by binding to specific
DNA sequences located in the regulatory regions of
target genes [40,41]. Receptors for androgens, atRA,
9cRA, and VD, have been shown to be present in
prostate tumor cells [3,4,7,9]. It remains unclear,
however, whether the mentioned ligands act directly
on genes related to cell cycle control or whether they
act indirectly by modulating the secretion of autocrine acting growth-promoting or growth-inhibiting
factors. The slowness of the effects rather suggests an
indirect mechanism of action.
The receptors for VD, (VDR), atRA (RAR), and
9cRA (RXR) share a family of similar hormone response elements configured as direct or everted repeats of the half-site consensus PuGGTCA. Both the
VDR and the RAR form heterodimers with the RXR
which can also function as an independent receptor
for 9cRA [42-471. In a number of systems synergistic
effects have been observed between 9cRA and the
ligand occupying the other half of the heterodimeric
receptor complex [48,49]. Such cooperative effects
could not be demonstrated in the LNCaP system. In
fact the proliferative andor antiproliferative effects of
VD,, atRA, and 9cRA were more or less additive.
Since the effects of 9cRA strongly resembled those of
atRA and since 9cRA binds with a higher affinity to
the RAR than to the RXR [50], it is quite possible that
the observed effects were mediated by the RAR.
In a number of systems retinoids, vitamin D, and
other proliferation inhibitors may induce terminal
differentiation. In HL-60 cells, e.g., RA induces a
granulocyte differentiation accompanied by growth
arrest [51]. Some data suggest that in prostate cancer
cells too differentiation therapy might result in phenotypic reversion and sustained growth arrest. PC-3
cells treated with PA for 1 week in culture, e.g., apparently loose the ability to form rapidly growing tu-
+
192
Esquenet et at.
0
-
e
inc. 3 d
”
60
-
45
-
i
I
-
I
U
0
4
m
p
30
-
U
0
5
E:
15-
-
P
72
77
2.
r
I
0
0
DCC-FCS
DCC-FCS
+
FCS
R1881
DCC-FCS
DCC-FCS
+
FCS
R1881
Fig. 6. Effect of prolonged pretreatment with VD, on LNCaP
cell proliferation in the presence and absence of androgens. LNCaP
cells were cultured for a period of I 2 days in the absence (open
bars) or in the presence of IO-* M VD, (hatched bars). Pretreated
cells were reseeded in 96-well plates a t a density of 5 x lo3 cells
per well and incubation (inc.) was continued for 3 or 6 days in
medium supplemented with 5% DCC-FCS. 5% DCC-FCS +
10- l o M R I88 I, or 5% complete FCS. A t the end of this period
[ ’HI-thymidine incorporation was measured. Results shown repreSD (n= 3) for a representative experiment.
sent the mean
mors in recipient athymic mice [lo]. Similarly, in a
clonal growth assay the effects of VD, and retinoids
on prostatic epithelial cells seem to be irreversible
[5,6]. The present data indicate that, at least in LNCaP cells, growth suppression by VD,, retinoids, or
PA is reversible. When cells pretreated for 6 or even
12 days with the mentioned growth inhibitors are reseeded at the same density (5 x lo3 cells/well in 96well multidishes) as untreated LNCaP cells, [,H]-thymidine incorporation and cell proliferation resume
within approximately 6 days. Further experiments
will be required to exclude that this is a specific property of LNCaP cells. It seems more likely, however,
that the reversible or irreversible character of the effects of the studied agonists depends on the selected
test conditions.
In summary, we demonstrated that each of the
substances tested could severely alter the growth responses of the LNCaP cell line both in the presence
and in the absence of androgens, and induce differentiated functions as measured by an increase in PSA
secretion. However, withdrawal of these substances
restored the initial growth response within 6 days.
Further studies using an in vivo model may be required to clarify the potential role of these compounds as therapeutic factors in limiting prostate cancer development.
Belgian National fund for Scientific Research
(N.F.W.O.). The excellent technical assistence of J.
Rosseels is kindly acknowledged.
ACKNOWLEDGMENTS
This research was supported by grant “Geconcerteerde Onderzoeksactie van de Vlaamse Gemeenschap”, grant 3.0015.88 from the Belgian National
Fund for Scientific Research (N.F.W.O.), and by a
grant from the Vereniging voor Kankerbestrijding.
Dr. J. Swinnen is a senior research assistant of the
*
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