(CANCER RESEARCH 50. 1966-1974. March 15, ]990|
Paracrine Stimulation of Polarized Secretion from Monolayers of a Neoplastic
Prostatic Epithelial Cell Line by Prostatic Stromal Cell Proteins1
Daniel Djakiew,2 Mary A. Tarkington, and John H. Lynch
Department of Anatomy and Cell Biology [D. D.], the Lombardi Cancer Research Center ¡D.D.J, and the Department of Surgery, Division of Urology [M. A. T.,
J. H. L.J, Georgetown University School of Medicine [D. D.J and Medical Center [M. A. T., J. H. L.J, Washington, DC 20007
ABSTRACT
binding sites are restricted to the mesenchyme, indicating that
morphogenesis entails an androgen-dependent proliferation of
stromal cells which in turn mediate epithelial proliferation and
development within the acini of the prostate (7). In addition,
the proliferation of epithelial cell growth within the prostatic
acini exhibits a regional heterogeneity, further indicating that
local control mechanisms, presumably through paracrine inter
actions with the stroma, regulate differentiation and develop
ment (8, 9). Implicit in the concept of stroma inducing, speci
fying, and maintaining epithelial phenotype and functional
activity is that disturbance of stromal-epithelial interactions
may lead to unregulated aberrant growth. In this context,
McNeal (10) suggested that benign prostatic hyperplasia in
adults may result from reversion of stroma to an embryonic
state leading to an aberrant stimulation of epithelial cell prolif
eration. Hence, detailed analysis of paracrine regulated growth
may eventually allow the development of strategies to modify
and manipulate aberrant prostatic proliferation.
By virtue of tight junctions between the apicolateral plasma
membranes of adjacent epithelial cells, prostatic acini are com
partmentalized and bathed in two distinct microenvironments,
one apical (luminal) and the other basal. The luminal microenvironment contains a variety of epithelial secretory products
including proteins such as prostatein (11), a spermine-binding
protein (12), a non-steroid-binding protein with a molecular
weight of 22,000 (13, 14), and a steroid-binding sialoglycoprotein (15). The basal surface of the epithelial cells rest on a
semipermeable basement membrane through which basally se
creted epithelial cell products may permeate, and in the oppo
site direction blood-borne constituents and stromal cell para
crine factors may also mix and permeate to form the basal
milieu. In order to study paracrine interactions between the
basal surface of epithelia and underlying stroma, as normally
occurs in vivo, we have adapted an in vitro experimental system
(16-18) devised to study other reproductive tract epithelia
(seminiferous epithelium, epididymis) to that of the prostate.
In this report we characterize the growth of confluent epithelial
sheets of a neoplastic prostate cell line (19) in bicameral cham
bers and report paracrine interactions between epithelial sheets
of the cell line and stromal cell secretory proteins derived from
prostatic primary cultures of young adult (50-day-old) rats.
The paracrine influence of prostatic stromal cell proteins on a neoplastic prostate cell line (PA-III) was investigated. We have utilized an
in vitro experimental model whereby confluent epithelial sheets of PAIII cells are grown on Matrigel-coated filters in bicameral chambers
(Millicell-HA). Confluence of the epithelial sheet was confirmed mor
phologically by electrical resistance measurements and by impedence of
| 'I l|inulin permeability across paracellular channels. Stromal cells were
isolated from the ventral prostate of 50-day-old rats by isopyknic Percoli
centrifugation. Purity (92%) of the isolated stromal cells was confirmed
by indirect immunofluorescence of vimentin intermediate filaments. Pros
tatic epithelial cells were negative for vimentin immunofluorescence.
Prostatic stromal cell secretory proteins with molecular weights >10,000
were placed in the basal reservoir of the bicameral chambers underneath
the confluent epithelial sheets of PA-III cells in a manner that mimics
the relationship between stroma and epithelia in vivo. After 24 h incu
bation the stromal cell proteins increased the (35S]methionine-Iabeled
protein secretion from the epithelial sheet of cells. Trypsinization of the
stromal cell secretory proteins eliminated the stimulatory effect on epi
thelial protein secretion. In addition, conditioned media from Swiss 3T3
fibroblasts, A431 cells, or bovine serum albumin did not stimulate epi
thelial protein secretion. Two-dimensional gel electrophoresis of
the |35S|methionine-labeled epithelial protein secretion showed that the
stromal cell proteins induced the secretion of a novel peptide (SE-1) from
the basal domain of the epithelial sheet of cells within the first hour of
metabolic labeling. These results indicate that stromal cell secretory
proteins contain a stimulatory protein that can induce overall protein
secretion as well as the vectorial secretion of a novel peptide from the
basal domain of PA-III epithelial cells. These results are consistent with
a paracrine interaction between epithelial and stromal cells in the regu
lation of prostatic secretion.
INTRODUCTION
The testosterone dependence of the prostate gland for the
development and maintenance of differentiated structure and
secretory function has long been recognized as a major mech
anism for the endocrine regulation of prostatic growth (1). In
addition to the global effects of testosterone, it has become
increasingly clear that both autocrine and paracrine factors
secreted by epithelial and stromal cells regulate localized pros
tatic growth. In this context, paracrine regulation of prostatic
growth was first suggested by Franks et al. (2) after observing
a lack of growth capacity of epithelia which had been separated
from its stroma. Subsequently, the elegant experiments of
Cunha et al. (3, 4) demonstrated that fetal mesenchyme
(stroma) induced prostatic epithelial morphogenesis from urothelium. Indeed, increased amounts of mesenchyme relative to
epithelium increased total prostatic growth (5), suggesting a
dose-dependent regulation of cellular proliferation. Prostatic
stroma and epithelia respond differentially to androgenic dep
rivation and stimulation (6). During development, androgenic
Received 12/29/88: revised 9/26/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This research was funded by NIH grant CA50229-01 (D. D.).
2 To whom requests for reprints should be addressed.
MATERIALS AND METHODS
Culture of Prostate Epithelial Cells. A Lobund Wistar rat epithelial
adenocarcinoma cell line (PA-III) was a kind gift from Dr. M. Pollard
(19). PA-III cells were propagated in 225-ml plastic tissue culture flasks
(Becton Dickinson Company, Lincoln Park, NJ) containing heat-inac
tivated 5% PCS3 (Hyclone Laboratories Inc., Logan, UT), DMEM/
Ham's F-12/Hepes buffer (Irvine Scientific, Santa Ana, CA), 2 HIM
glutamine (Sigma Chemical Co., St. Louis, MO), 100 units/ml penicil
lin (Sigma), 100 Mg/ml streptomycin (Sigma), and IO"7 M testosterone
'The abbreviations used are: PCS, fetal calf serum; DMEM, Dulbecco's
minimum essential medium: Hepes, 4(2-hydroxyethyl)-l-piperazineethanesulfonic acid: SFDM. serum-free defined medium: PBS. phosphate-buffered saline:
SCCM. stromal cell-conditioned medium.
1966
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1990 American Association for Cancer Research.
PARACRINE STIMULATION OF PROSTATE SECRETION
(Sigma). All cultures were maintained in a humidified incubator at
37°Cin 5% CU2/air. Culture media was changed every third day.
Subsequently, PA-III cells were removed from the culture flasks with 5
mg/ml trypsin and 2 mg/ml EDTA in balanced salt solution without
calcium and magnesium (Whittaker Bioproducts, Walkersville, MD).
The resulting single-cell suspension of PA-III cells was washed by
centrifugaron at 200 x g in culture media and 5 x 10*cells seeded on
basement membrane Matrigel (Collaborative Research Ine, Lexington.
MA) coated Millipore filters (0.45-^m pore size) in bicameral (MillicellHA) dual compartment chambers (Millipore Corp.. Bedford, MA). A
schematic diagram of epithelial cells cultured in the bicameral chambers
is shown in Fig. 1. PA-III cells grown in the bicameral chambers were
cultured in either 5% PCS (as above) or a SFDM consisting of 2 Mg/
ml zinc-free insulin (Collaborative Research), 10 ng/ml epidermal
growth factor (Collaborative Research), l ¿ig/mltransferrin (Sigma).
10~7M hydrocortisone (Sigma), 2 HIMglutamine (Sigma), and 10~7 M
testosterone (Sigma) formulated in DMEM/Ham's F-12/Hepes (Irvine
Scientific) containing 100 units/ml penicillin (Sigma) and 100 Mg/ml
streptomycin (Sigma).
Characterization of Confluence. Electrical resistance to the passage
of current across the monolayers of epithelial cells grown in the bica
meral chambers was measured using an epithelial voltohmmeter (World
Precision Instruments Inc., New Haven, CT) as an indication of the
establishment of transepithelial permeability barriers, as previously
described (17). Background electrical resistance measurements on Matrigel-coated filters in the absence of PA-III cells were subtracted from
measurements of the epithelial cell resistance.
| 'I l|Imilin permeability across Matrigel-coated filters in the absence
or presence of PA-III cells was determined by placing 1 x IO6 cpm of
[ 'HJinulin (ICN Radiochemicals, Irvine, CA) in the basal reservoir of
the bicameral chambers. Subsequently, over a 6-h period, an aliquot of
culture media from the apical reservoir was analyzed for the content of
['Hjinulin in a liquid scintillation counter (Beckmann LS 8000; Wakefield, MA).
Morphology of PA-III cells grown in bicameral chambers was as
sessed by electron microscopy as previously described (20). Confluent
growth of PA-III cells in the bicameral chambers was confirmed by
staining some of the epithelial sheets that had been fixed for electron
microscopy with 4.8 g/liter Harris' alum hematoxylin (Accra Lab,
Bridgeport, NJ), followed by rinsing in PBS, substitution with a graded
series of ethanols, and clearing the cells in xylene. The epithelial sheets
were then attached to microscope slides with tissue tack (Polysciences,
Warrington, PA) and mounted in Flo-texx (Lerner Labs, Stanford, CT)
for observation and photography.
Isolation of Stremai Cells and Preparation of Conditioned Media. The
ventral prostate from 50-day-old Wistar rats that had been carbon
dioxide asphyxiated were cleaned of associated adipose tissue and
minced into small (1-3 mm1) blocks of tissue with a pair of fine scissors
PROSTATE BICAMERAL CULTURE CHAMBER SYSTEM
Apical
•¿
',
h
' "
'U'"'
-^—
' ' ""J
reservoir
^Epithelial cells
*'
"
'"'•f.tf
l t M
l t ltt:l
-"-
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Ut
•¿
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in DMEM/Ham's F-l 2/Hepes culture media. The blocks of tissue were
transferred into 20 ml of a filter-sterilized enzymatic digestion medium
consisting of 0.3 Mg/ml DNase I (Sigma), 1.0 mg/ml collagenase
(Gibco), and 1 mg/ml hyaluronidase (Sigma). During enzymatic diges
tion, the tissue was placed on a shaking water bath (100 cycles/min) at
37°Cand periodically aspirated in a 10-ml pipet. Dissociation of the
stroma from the epithelial acini was monitored under an inverted phase
contrast microscope. After substantial separation of acini from stroma,
the acini were allowed to sediment at unit gravity in a 50-ml centrifuge
tube, and the stromal cell enriched supernatant was aspirated, centrifuged at 200 x g for 3 min and the pellet resuspended in 20 ml of a
second digestion media consisting of 0.07 mg/ml Dispase (Boehringer
Mannheim GmbH, West Germany) and 0.3 Mg/ml DNase I (Sigma).
This stromal cell-enriched preparation was placed on the shaking water
bath and monitored until a single-cell suspension was obtained. The
single-cell suspension was washed in 50 ml culture media by centrifugation at 200g for 3 minutes, resuspended in 2 ml culture media and
further separated on preformed continuous isopyknic Percoli (Phar
macia, Uppsala, Sweden) gradients according to the method of Orlowski et al. (21). Formation of the Percoli density gradient was
confirmed using colored density marker beads (Pharmacia). The pros
tate cell fractions were aspirated, and an aliquot of each fraction was
plated on glass coverslips in 5% FCS supplemented with IO"7 M
testosterone. The stromal cell fractions were washed in culture media
by centrifugation and incubated in 225-ml culture vessels containing
5% FCS supplemented with IO"7 M testosterone.
After 2 days of culture, the stromal cells were washed for 30 min
three times with DMEM/Ham's F12/Hepes/antibiotics supplemented
with 10~7 M testosterone. The stromal cells were then cultured in the
former media for 24 h, after which the stromal cell-conditioned media
was collected and centrifuged at 10.000 x g and the supernatant
immediately frozen at —¿20"C.
This procedure was repeated on subse
quent days to collect additional stromal cell-conditioned media.
Stromal cell-conditioned media was concentrated and desalted with
distilled water by centrifugation in Centriprep-10 concentrators (Amicon, Danvers, MA). Subsequently, the concentrated proteins in the
Centriprep-10 retained material were lyophilized. As cellular controls
secretory proteins from Swiss 3T3 fibroblasts and A431 cells were
prepared in a manner similar to the stromal cell-conditioned media.
Immunocytochemistry. Prostatic cells separated on isopyknic Percoli
gradients and cultured on glass coverslips were screened for vimentin
intermediate filaments by indirect immunofluorescence. Methanolfixed cells were blocked for 60 min with 3% ovalbumin in PBS at 37°C
and incubated with goat anti-murine vimentin antibody (1:100; Miles
Laboratories, Naperville, IL) at 37°Cfor 60 min. The cells were washed
three times in PBS. then incubated at 37°Cfor 60 min in rhodamineconjugated rabbit anti-goat IgG (1:100; Kirkegaard and Perry Labora
tories. Gaithersberg. MD). Subsequently, the cells were washed three
times in PBS, mounted, and viewed with a Zeiss photomicroscope fitted
with an epifluorescence attachment.
Culture of PA-III Epithelial Monolayers with Stromal Cell Proteins.
The stromal cell protein lyophilate was resuspended in a small volume
of methionine-deficient DMEM and the protein concentration meas
ured with the Bio-Rad protein assay (Bio-Rad, Richmond. CA). Con
fluent epithelial sheets of PA-III cells, grown in bicameral chambers,
were placed in 24-well cluster tissue culture plates and 400 n\ SCCM
containing 100 fiC\/m\ [15S]methionine (ICN, Irvine, CA) was placed
in the basal reservoir, while 400 n\ methionine-deficient SFDM were
placed in the apical reservoir. The epithelial sheets were incubated with
the SCCM at 37°Cin 5% CO2/air. As controls the SCCM was substi
tuted with conditioned media from Swiss 3T3 fibroblasts, A431 cells,
or bovine serum albumin. For the time course studies the metabolically
labeled PA-III epithelial cell secretory proteins were collected over a
period of 24 h. The epithelial cells were digested in l N NaOH and
Stremai cells or conditioned medium
neutralized with an equal volume of l N HC1. Subsequently, secreted
and intracellular [15S]methionine-labeled protein was precipitated with
Fig. 1. A schematic diagram of a bicameral culture chamber used to grow PAIII epithelial cells at confluence on a Millipore Tiller coated with extracellular
TCA (65). For the dose-dependent studies PA-III cells were incubated
matrix. By virtue of the confluent epithelial sheet of cells culture media in the
with 3, 15, and 30 Mg/ml SCCM over 24 h. In order to degrade protein,
apical and basal reservoirs are prevented from mixing, thus compartmentalizing
an aliquot of the 30 Mg/ml SCCM was trypsinized and subsequently
the chambers. Stromal cells or their conditioned media are included in the basal
reservoir in a spatial relationship comparable to their in viro location.
neutralized with soybean trysin inhibitor according to the method of
1967
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1990 American Association for Cancer Research.
PARACRINE STIMULATION OF PROSTATE SECRETION
Djakiew
initially
quently,
min and
and Dym (22). For the pulse-chase studies PA-III cells were
incubated for 24 h with or without 30 Mg/ml SCCM. Subse
the cells were pulsed with 1.5 mCi/ml ["S]methionine for 20
chased with a 1000-fold excess of [32S)methionine. All collected
H,.
"•
¿..
protein samples were made 2 mM with phenymethylsulfonyl fluoride,
and the secretory proteins were centrifuged at 10,000 x g for 30 min.
Except for the time course studies, unincorporated [35S]methionine was
removed from the supernatent of the samples by repeated dilution (8
times) in distilled water followed by centrifugation of the metabolically
labeled proteins in Centriprep-10 microconcentrators (Amicon). Total
[35S]methionine-incorporated activity was determined by resuspending
the Centriprep-10 retained material in distilled water to its original
volume, or the trichloroacetic acid precipitate in 100 n\ 0.1 N NaOH,
and measuring ["SJmethionine activity in a liquid scintillation counter
(Beckmann). The filters on which the epithelial cells were growing were
removed from the bicameral chambers, and the DNA content of the
cells was estimated according to the method of Burton (23). For the
time course studies DNA was estimated from parallel cultures similarly
treated with or without SCCM. Total DNA/bicameral chamber was
converted to cell number using a previously determined estimate of
13.0 ±0.4 Mg(SEM) DNA/106 PA-III epithelial cells. All estimates of
["S]methionine-labeled protein secretion were normalized to the total
number of PA-III cells in each bicameral chamber.
Electrophoresis and Fluorography. Protein samples were prepared
for two-dimensional polyacrylamide gel electrophoresis according to
the method of O'Farrell (24) as modified by Djakiew and Dym (22).
Briefly, protein samples were diluted in an equal volume of nonreducing
sample buffer and heated to 100'C for 90 s. Equal cpm of protein
samples were separated on 4% polyacrylamide tube gels (3 mm inside
diameter) by isoelectric focusing over 6900 V-h. Proteins were separated
in the second dimension on polyacrylamide slab gels with 4% stacking
gels and 10% separating gels. Standard peptides for determining isoe
lectric point in the first dimension and molecular weight in the second
dimension were obtained from Bio-Rad and New England Nuclear
(Boston, MA), respectively. Fixed gels were impregnated with fluors by
soaking twice in dimethyl sulfoxide for 30 min each, followed by 3 h in
22% PPO/dimethyl sulfoxide and washing in distilled water for 1 h.
Gels were dried under vacuum at 50°Cand exposed for autoradiography
to X-Omat AR diagnostic film (Kodak, Rochester, NY) for 4 days at
-70°C.
Statistical Analysis. The statistical significance of differences between
treatments was tested by analysis of variance using the Newman-Keuls
multiple range test and by Student's t test. The SEM values that are
given as estimates of the dispersion in the results were calculated for
each treatment from estimates of the variance between replicates.
RESULTS
Characterization of Confluence of the Epithelial Cell Monolayers in the Bicameral Chambers. Fig. 2 shows a low power
view of PA-III epithelial cells that have been removed from a
bicameral chamber and stained with Harris' alum hematoxylin.
•¿vx.'.Ã-Cx
•¿
âA^:'«':'
&îM
.., ;
':--:..
,;*'*••
-v'•:,".
?l>- •¿â€¢.•.-.-.•:••.••
:
^ .
„¿v>
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Fig. 2. Light micrograph of PA-III epithelial cells grown to confluence on the
surface of a Millipore filter which has been removed from the bicameral chamber
and stained with Harris' alum hematoxylin. Bar, 86 ¡mi.
cells grown in SFDM were columnar (Fig. 3/1), whereas those
cells grown in 5% PCS were relatively squamous (Fig. 3Ä).
Fig. 4 shows the electrical resistance of PA-III epithelial cells
seeded in the bicameral chambers and cultured in either SFDM
or 5% FCS. The electrical resistance of the forming epithelial
sheets of PA-III cells differed significantly (P < 0.05) when
grown in either SFDM or 5% FCS. Irrespective of the culture
media, the peak electrical resistance of the epithelial sheets
occurred on approximately the 5th day of culture. In the case
of epithelia grown in SFDM, the electrical resistance remained
relatively constant for an additional 7 days in culture, whereas
the electrical resistance of epithelia grown in 5% PCS gradually
declined over the following 7 days in culture.
Fig. 5 shows the passage of | 'lljinulin across bicameral
chambers (basal to apical) in the absence of epithelial cells or
across confluent epithelial sheets of cells grown for 5 days in
either SFDM or 5% FCS. The passage of [3H]inulin across the
bicameral chamber in the absence of any cells was approxi
mately 2.8% of the total added/h. In contrast, the rate of
passage of [3H]inulin across a confluent culture of PA-III cells
grown in 5% PCS was approximately 0.5% of the total added/
h, whereas the rate of passage [3H]inulin across a confluent
culture of PA-III cells grown in SFDM was approximately
0.23% of the total added/h.
Separation of Stromal Cells from Epithelial Cells. Fig. 6, A
and A, show a phase contrast image and the corresponding
indirect immunofluorescence of vimentin intermediate fila
ments, respectively, of a primary culture of stremai cells which
have been separated on continuous isopyknic Percoli gradients
and grown on glass coverslips. The stromal cells (Fig. 6/4)
consisted of attenuated and spindle-shaped cells. The cytoplasm
of these cells contained numerous stress fibers and was deficient
in vesicular inclusions. For comparison a primary culture of
epithelial cells that had been similarly prepared is shown in
Fig. 6, C and D. The phase contrast image of the epithelial cells
(Fig. 6C) exhibited numerous prominent refractive vesicles in
the cytoplasm. Note that vimentin intermediate filament im
munofluorescence was not detected in the epithelial cell culture
(Fig. 6D). Based on vimentin immunofluorescence the purity
of the stromal cells isolated by isopyknic sedimentation was
92 ±3% (n = 3).
Stimulation of PA-III Epithelial Cell Protein Secretion by
Stromal Cell-conditioned Media. Figs. 7 and 8 show the time
course of [35S]methionine incorporation into intracellular and
Note that the epithelial sheet of cells has grown to confluence
over the surface of the filter. Fig. 3, A and B, show low power
photographic montages of epithelial cells grown for 5 days to
confluence on the filters in either SFDM or 5% PCS, respec
tively. The characterization of the PA-III adenocarcinoma cell
line (19) and its ultrastructural characteristics (25) have been
discussed in the literature, hence only pertinent features of
these cells grown in the bicameral chambers will be described.
In this context, it is noteworthy that, irrespective of the culture
conditions, the epithelia were lined by short microvilli on their
apical surface (Fig. 3, A and B) and were joined at their apicallateral plasma membranes by tight junctions. In addition, the
basal cytoplasm of the cells interdigitated into the pores of the
Millipore filters thereby enhancing the basal surface area of the
cells. The most pertinent morphological difference between
epithelia grown under different culture conditions was that the
1968
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1990 American Association for Cancer Research.
PARACRINE STIMULATION
OF PROSTATE SECRETION
Fig. 3. Photographic montages of PA-II1
epithelial cells grown to confluence in bica
meral chambers and cultured in either SFDM
for 5 days (A) or in 5% PCS for 5 days (B).
Note the cuboidal to columnar morphology of
the cells grown in SFDM (A) compared to the
cells grown in 5% PCS which appear morpho
logically squamous (B). Bars, 5.5 ».in.
cpm/106 cells), trypsinization and subsequent neutralization of
the 30-Mg/ml SCCM" treatment eliminated the stimulatory
•¿
SFDM
»
5% PCS
1000
» 500
567
Time (days)
8
9
10
11
12
13
Fig. 4. Electrical resistance of PA-III epithelial cells over a 13-day duration.
Note that the electrical resistance of PA-III cells grown in SFDM was significantly
greater (P < 0.05) than PA-III cells grown in 5% PCS. Results are expressed as
the means ±SE (bars) (n = 4).
216 '
Fig. 5. Percentage of |'H]inulin passing from the basal to the apical reservoirs
of the bicameral chambers over a period of 6 h through a filter coated with
Matrigel (no cells) and through confluent epithelial sheets of PA-III cells cultured
in either 5% PCS or SFDM.
secretory protein, respectively, of PA-III cells incubated with
or without 30 Mg/ml SCCM. ["S]Methionine incorporation
into ¡nirat-elliilarand secretory protein was saturated by 18 h
for both the control and SCCM treatments. Whereas ["S]
effect of the SCCM on PA-III epithelial protein secretion (98
±11%, n = 3). In addition, percent changes in [li;S]methioninelabeled protein secretion from the PA-III cells incubated with
30 Mg/ml bovine serum albumin (94 ±6% of control values, n
= 3) or conditioned media from 3T3 fibroblasts (101 ±7% of
control values, «= 3) and A431 cells (93 ±8% of control
values, n = 3) were not significantly different from the control
cultures.
Stromal Cell-conditioned Media Stimulation of Polarized Pro
tein Secretion from PA-III Cell Monolayers. Polarity of [15S]
methionine-labeled protein secretion into the apical reservoir
(Fig. 10, A, C, and E) and basal reservoir (Fig. 10, B, D, and F)
under control conditions (Fig. 10, A and B) and when 30 pg/
ml cold (nonlabeled) SCCM was added to the basal reservoir
(Fig. 10, C and D) or the apical reservoir (Fig. 10, E and F)
was investigated by two-dimensional polyacrylamide gel electrophoresis. In addition to SCCM stimulating a quantitative
increase in [•"S]methionine-labeled protein secretion (Fig. 9)
from PA-III cells, SCCM (30 Mg/ml) also induced the exclusive,
basally directed secretion of a novel peptide, designated SE-1,
from PA-III cells (Fig. 10, D and F) relative to control (nonstimulated) monolayers of PA-III cells (Fig. 10, A and B). This
peptide (SE-1) has an isoelectric point of approximately 5.6,
and an apparent molecular weight of approximately 95,000.
This peptide was absent from the apical (Fig. 10A) and basal
(Fig. 10B) secretions of control cultures, as well as from the
apical secretions of SCCM-stimulated PA-III monolayers (Fig.
10, C and E). Furthermore, the stimulation of the basally
directed secretion of peptide SE-1 occurred irrespective of
whether the SCCM was added to the basal reservoir (Fig. 10,
C and D) or the apical reservoir (Fig. 10, E and F) of the
bicameral chambers. Pulse-chase studies of PA-III cell secretion
into the basal reservoir indicated that 50% of the protein was
secreted in 36-43 min (Fig. 11). Peptide SE-1 was secreted
within the first hour (Fig. \2A) and could not be seen on twodimensional gels at subsequent times of the chase period (not
shown). In control cultures not stimulated by SCCM, peptide
SE-1 was not secreted during the first hour (Fig. \2B) or at
later times of the chase period (not shown).
methionine incorporation into intracellular proteins was not
significantly different between the two treatments (Fig. 7),
SCCM stimulated (P < 0.05) protein secretion from the PAIII cells over the 24-h incubation period (Fig. 8). Fig. 9 shows
that SCCM, placed in the basal reservoirs of bicameral cham
bers, significantly (P < 0.05) stimulated [15S]methionine-labeled
protein secretion from the confluent epithelial sheets of PA-III
DISCUSSION
cells in a dose-dependent relationship over a period of 24 h.
When [15S]methionine-labeled protein secretion was expressed
After the growth of PA-III cells in bicameral chambers for 5
as a percentage relative to the control value (100% = 3231 days the sheets of epithelial cells appeared morphologically
1969
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PARACRINE STIMULATION OF PROSTATE SECRETION
Fig. 6. Prostatic cells isolated from the ven
tral prostate of SO-day-old rats and separated
by isopyknic Percoli centrifugation. I and B,
phase contrast image of stremai cells and the
corresponding indirect immunofluorescenceof
»imrniniintermediate filaments, respectively.
C and I), phase contrast image of epithelial
cells and the corresponding lack of vimentin
immunofluorescence, respectively. Bars, 4.4
| 3
o
a. 2
I
Q>
1
U
12
24
18
Time (hrs)
Fig. 7. Time course of ["S]methionine incorporation into PA-III cellular
protein (cpm x lO'/'O' cells) in the absence (control) or presence of 30 fig/ml of
SCCM placed in the basal reservoir of the bicameral chamber. Results are
expressed as the means ±SE (bars) (n= 3).
r
o
<Ã-4
•¿2
12
Time Chrs)
Fig. 8. Time
secretor) protein
»ig/mlof SCCM
are expressed as
24
course of [3!S]methionine incorporation into total PA-III cell
(cpm x IOJ/10* cells) in the absence (control) or presence of 30
placed in the basal reservoir of the bicameral chambers. Results
the means ±SE (bars) (n = 3).
E 2
w1
Fig. 9. Dose-dependent influence of SCCM (jig/ml) on |"S]methionine-Iabeled protein secretion from PA-III cells. Protein secretion is expressed as a fold
increase relative to control cultures ¡nthe absence of SCCM. Results are expressed
as the means ±SE (bars) (n = 3).
confluent at the light (Fig. 2) and electron microscopic levels
(Fig. 3, A and B). Concurrently, after the same period, the
sheets of epithelial cells were characterized by a maximal elec
trical resistance. Electrical resistance has been widely used as
an indicator of the formation of transepithelial permeability
barriers (26) across cell lines of the canine kidney (27, 28),
porcine kidney, toad urinary bladder (26), and human intestine
(29) as well as in primary cultures of murine mammary epithe
lium (30) and rodent Sertoli cells (17). Since tight junctions are
the rate-limiting permeability barrier of the paraceli ular channel
across a confluent sheet of epithelial cells (31), the degree of
electrical resistance has been correlated with the number of
strands in the occluding junctions between adjacent cells (29).
Indeed, differences in the electrical resistance of PA-III epithe
lial sheets grown in SFDM or PCS were paralleled by differ
ences in the transepithelial flux of the extracellular space
marker, [3H]inulin. Hence, based on the morphological obser
vations, electrical resistance measurements, and [3H]inulin
permeability studies, we conclude that PA-III cells grown to
confluence in bicameral chambers and cultured, particularly in
SFDM, prevent the mixing of fluids between the apical and
basal reservoirs of the bicameral chambers. Indeed, these results
are consistent with a compartmentalization
of the milieu sur
rounding the epithelial monolayer into separate apical and basal
domains, as normally occurs in vivo, thereby validating the
prostatic bicameral chamber model for the examination of
polarized epithelial cell secretion and paracrine interactions
with underlying stroma or stromal cell-conditioned media.
The cytoplasm of vertebrate cells contain, in addition to
microtubules and microfilaments, a third class of filamentous
structures called intermediate filaments. Five major classes of
intermediate filaments have hitherto been characterized both
biochemically and immunologically (32). They are glial fila
ments found in glial cells; neurofilaments found in neurones;
desmin filaments found predominantly in smooth, skeletal, and
cardiac muscle cells; keratin filaments found in epithelial cells;
and vimentin filaments found predominantly in mesenchymal
cells (32, 33). The broad cross-reactivity of vimentin antibodies
with mesenchymal cells of mammalian, avian, and amphibian
1970
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PARACRINE STIMULATION OF PROSTATE SECRETION
-92.5
-92
-69
-69
-46
-46
5
i
Fig. 10. Two-dimensional
gel auloradiographs of ["S]mcthionine-labeled proteins se
creted from the apical (A, C, E) and basal (B,
D, F) surfaces of PA-III epithelial cells grown
at confluence in bicameral chambers for 24 h.
Control cultures (A, B) were grown in SFDM.
Stremai cell conditioned media (30 ng/nil)
added to the basal reservoir (C, D) or apical
reservoir (E, F) of the bicameral chambers
stimulated the exclusively basal secretion of
the novel peptide SE-1 (D, F). Peptide SE-1
was absent from the corresponding apical se
cretions (C, E). Ordinate*, molecular weight
(X10~3); abscissas, isoelectric point. Autoradi-
-92.5
-69
-46
-46
ographs are representative of 5 independent
experiments, n = 5.
5
SE-1
-92.5
-69
-69
-
-46
5
i
f
.E 15
-46
•¿
Cumulative
•¿
Chase
10
12
Time (hrs)
Fig. 11. Pulse-chase of |"S|methionine-labeled protein secretion (cpm x IO4/
10* cells) from the basal surface of PA-III cells stimulated by 30 fig/ml SCCM
over a period of 12 h. Cultures were pulsed with 1.5 mCi/ml of ["S|methionine
for 20 min and chased with a 1000-fold excess of [32S]methionine for up to 12 h.
Secreted protein was collected at the indicated times. Results are representative
of 3 experiments.
origin indicates that this protein is well conserved across the
various phylogenetic classes (34-36). In this context, the pri
mary antibody against murine vimentin used for indirect immunofluorescence readily cross-reacted with the rat prostatic
stromal cells while not detecting any appreciable vimentin in
the epithelial cells. Sherwood et al. (66) also found prominent
f(L
expression of vimentin in prostatic stromal cells isolated from
human benign prostatic hyperplasia tissue, with a very low level
of expression in epithelial cells. Hence, we were able to use
vimentin localization as a method of distinguishing between
the rat stromal and epithelial cells. Following isopyknic Percoli
centrifugation of the enriched stromal cell preparation of pros
tatic cells, we obtained a stromal cell fraction of 92% purity.
This purity is comparable to that obtained by other workers
(21, 37) who have used isopyknic sedimentation techniques to
separate prostatic stroma from epithelial cells and have subse
quently characterized the purity of their cell separation by the
exclusive secretion of prostatic acid phosphatase by the epithe
lial cells. As a means of distinguishing between cell types within
prostatic tissue, vimentin intermediate filament localization
within stromal cells has several advantages over measuring
epithelial prostatic acid phosphatase secretion. In this regard,
vimentin is an extremely stable cytoskeletal component (38),
whereas prostatic acid phosphatase is highly labile (39), requir
ing the addition of compounds such as citric acid to stabilize
the enzyme. In addition, the expression of stromal vimentin is
stable in vitro, even under serum starvation conditions (40),
whereas the secretion of epithelial prostatic acid phosphatase
1971
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1990 American Association for Cancer Research.
PARACRINE STIMULATION
SE-1
-92.5
Fig. 12. Two-dimensional
gel autoradiographs of ["S|methionine-labeled protein se
creted into the basal reservoir following a 20min pulse and chase with a 1000-fold excess
of ("S|methionine. Peptidc SE-1 was present
after a 1-h chase in cultures stimulated with
30 ,.L' ml SCCM (.4) but was absent in control
cultures (B). Ordinales, molecular weight
(xlO~J); abscissas, isoelectric point. Results
are representative of 3 experiments.
OF PROSTATE SECRETION
-92.5
-69
-69
-46
-46
5
i
rapidly declines in vitro (41). Hence, we have used vimentin
localization as a means of confirming the purity of our stromal
cell preparation, thereby allowing us to use stromal cell-condi
tioned media to investigate paracrine interactions between prostatic stroma and confluent sheets of PA-III epithelial cells.
The inductive influence of mesenchymal cells on the expres
sion of epithelial cell phenotype during the embryonic morpho
genesis of specific tissues has become a fundamental concept in
developmental biology. Mesenchymal-epithelial
interactions
have been demonstrated to regulate fetal organogénesisof mam
mary tissue (42, 43), epidermal appendages such as hair and
feathers (44,45), and the prostate gland (3), to name a few. The
concept of stromal-epithelial interactions regulating fetal dif
ferentiation and development has also been extended to post
natal tissue remodeling where paracrine interactions between
adjacent dissimilar cell types are considered to regulate local
ized growth. In this regard, the morphogenetic development of
mammary tissue during pregnancy is considered to be a hor
mone-dependent epithelial-stromal regulated growth process
(46, 47). Furthermore, the process of spermatogenesis is an
example of ongoing growth and differentiation in the postnatal
environment (48) where germ cells can induce the de novo
expression of Sertoli cell secretory proteins in a paracrine
regulatory manner (22). In this report we demonstrate that a
trypsin-sensitive factor(s) with a molecular weight > 10,000 in
stromal cell-conditioned media stimulates PA-III epithelial cell
secretion in a dose-dependent manner consistent with paracrine
regulation of rat prostatic growth. Indeed, the preliminary
results of Sherwood et al. (49) demonstrating stimulation of
human prostatic epithelial cell growth by human stromal cell
secretory proteins are comparable to the work reported herein.
In addition, Swinnen et al. (67) recently demonstrated a factor
in rat prostatic SCCM which could stimulate Sertoli cell secre
tion. Although several systemic growth factors such as insulin
(50-52), epidermal growth factor (51), and prostatropin (53,
54), as well as the autocrine prostate-derived growth factor (63,
64), are known to stimulate prostatic growth, the identity of
the stromal cell-secreted paracrine protein(s) remains to be
elucidated. Since live stromal cells were not required to stimu
late PA-III epithelial cell secretion, the observed interaction
was neither dependent on cell to cell contact nor due to the
deposition of extracellular matrix by the stroma. Nevertheless,
since the PA-III cells were seeded on Matrigel, the presence of
a suitable substratum may be a permissive requirement for
subsequent stromal stimulation of epithelium, as has been
demonstrated for mammary tissue (55). Indeed, the observa
tions that stromal stimulation of mammary epithelial cell se
cretion may be exerted from multiple stromal cell types (56)
present the possibility that the diverse population of stromal
7—
i
cells within the prostate may secrete different stimulatory fac
tors and that this diverse population of stromal cells may
differentially regulate epithelial secretion and growth during
development and in the postnatal environment.
Many differentiated cells are morphologically polarized and
express secretory proteins in a vectorial manner consistent with
a domain-specific interaction with the surrounding milieu and/
or adjacent cells. In vitro, vectorial secretion by Madin-Darby
canine kidney cells (57, 58), mammary epithelium (59), and
Sertoli cells (22, 60) is considered to reflect compartmentalization of tissue function of the in vivo counterparts of these
cells (18). In the rat prostate our work shows that the stromal
cell protein(s) induced the vectorial secretion of a novel peptide
(SE-1) exclusively from the basal surface of the neoplastic PAIII epithelial cells. Curiously, the induction of peptide SE-1
secretion occurred irrespective of whether the stromal cell pro
teins were added to the apical or basal surface of the PA-III
cells. This could be due to the missorting and aberrant expres
sion of corresponding receptors on the apical plasmalemma of
the transformed PA-III cells. Alternatively, the stromal cell
proteins may have been endocytosed from the apical surface of
the PA-III cells, nonspecifically (fluid phase), and following
internalization
stimulated peptide SE-1 expression. Even
though the stimulation of peptide SE-1 secretion was not do
main specific, the secretion of peptide SE-1 was domain spe
cific. Hence, the stromal cell protein induction of peptide SE1 secretion from the basal domain of the PA-III epithelial cells
in the direction where stromal cells would normally reside in
vivo is consistent with a compartmentalized paracrine interac
tion between prostatic epithelia and stroma in the regulation of
prostatic secretion. In this context, it would be of interest to
determine whether the stromal cell protein induction of peptide
SE-1 secretion from epithelial cells normally occurs in vivo, in
which case the peptide could be used as a marker of paracrine
regulated growth. Considering the variable and often unpre
dictable fluctuations in the secretion of prostate-specific antigen
and prostatic acid phosphatase used as serum markers of ma
lignant carcinomas (61, 62), measurement of an epithelial pro
tein such as peptide SE-1 which is basally secreted, and there
fore may occur in serum, may prove to be a more reliable and
specific marker for the early diagnosis of malignant prostatic
carcinomas.
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. , ,. . „¿
.
,
, . .
. „¿
., ... . ,_ .„, ...
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1974
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Paracrine Stimulation of Polarized Secretion from Monolayers of
a Neoplastic Prostatic Epithelial Cell Line by Prostatic Stromal
Cell Proteins
Daniel Djakiew, Mary A. Tarkington and John H. Lynch
Cancer Res 1990;50:1966-1974.
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