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Institute of Reproduction and Development (S.J.M., S.L.M., H.W., G.P.R.), 27–31 Wright St, Clayton, Victoria, Australia, 3168; Department of Biological and Molecular Sciences (L.W.E., N.P.G.), Oxford Brookes University, Headington, Oxford, OX3 0BP, United Kingdom
Address all correspondence and requests for reprints to: Associate Professor G. P. Risbridger, Institute of Reproduction and Development, 27–31 Wright Street, Clayton, Victoria, Australia 3168. E-mail: gail. risbridger{at}med.monash.edu.au
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Introduction |
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We have reported that activin and FS are expressed and localized to human prostate tissue from men with high grade cancer (13), and the expression of mRNAs for the activin subunits and the activin receptors has been recorded in the commonly studied human prostate tumor cell lines, LNCaP, DU145, and PC3 (14, 15, 16, 17, 18). One of these lines, the LNCaP tumor cells, is responsive to activin A and growth inhibition occurs by inhibiting cell proliferation and inducing apoptosis (14, 15, 19). However, this response is not manifested by the PC3 cells, which are insensitive to activin A; the response by the DU145 cells to activin A is intermediate (14). The action of activin ligands occurs through the activin receptors that are known to be expressed in all three tumor cell lines. The relative levels of the receptor mRNAs or proteins may differ between cells or, as in the case of colon cancers that are resistant to TGFß, the receptors may be mutated and inactive (20). However, before receptor binding and signaling, access of activin A ligand to receptors may be regulated through the activin binding protein follistatin. We have also reported that the expression of FS by the three cell lines is different; all three lines expressed FS315 mRNA, but only the PC3 cells had detectable expression of FS288 mRNA (14). These observations have led us to postulate that the differential response to activin A is due, in part, to a different pattern of FS protein expression. Until recently, it has not been possible to test this hypothesis, but the development of new, specific antibodies for the detection of FS288 and FS315 by immunostaining (10, 13) and the development of an enzyme-linked immunosorbent assay (ELISA) for the measurement of FS288 (21), now makes these studies possible. Thus, the aims of this study were to compare the localization of the FS proteins to LNCaP and PC3 cells by immunohistochemistry and Western blot analysis, and to measure the production of activin A and FS288 by specific ELISA.
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Materials and Methods |
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Growth factors and other reagents
Human recombinant activin A was kindly provided by Biotech
Australia Pty. Ltd. (East Roseville, New South Wales, Australia). Human
recombinant follistatin 288 (hrFS288) was obtained from the National
Hormone and Pituitary Program (Rockville, MD). Activin A was stored
freeze-dried in 0.1% trifluoroacetic acid/acetonitrile and
reconstituted in DMEM for use in cell cultures. FS288 was stored freeze
dried at -70 C. For use in ELISAs FS was reconstituted in 1 x
PBS + 0.1% BSA (Sigma, St. Louis, MO) and stored at -20
C in aliquots of 500ng/ml. Mouse IgG1 monoclonal antiserum was obtained
from DAKO Corp. A/S (Glostrup, Denmark) and used as a
negative control. Tritiated thymidine (3H-thymidine) was
purchased from NEN Life Science Products (Boston, MA).
Human recombinant follistatin 315 (hrFS315) was kindly provided by Professor H. Sugino (Institute for Enzyme Research, Tokushima, Japan).
Follistatin antibodies
A monoclonal antibody (17/2) to human recombinant FS288 was
raised by Evans et al. (21) and has previously been used for
the detection of FS288 by an ultra sensitive 2-site ELISA in pregnant
female human serum (21). The same antibody has also been used for
localization of FS288 by immunohistochemical methods (10).
The monoclonal antibody H10 was produced in Balb/c mice against a synthetic peptide (sequence: C-D-E-D-Q-D-Y-S-F-P-I-S-S-I-L-E-W) corresponding to the C-terminus of human FS315 (22) using a procedure previously described by Groome et al., 1996 (23). The specificity of the purified antibody was tested by a screening ELISA in which purified antibodies were titrated across ELISA plates coated with hrFS288, or hrFS315, H10 Ab showed immunoreactivity to hrFS315 but not to hrFS288, indicating the H10 Ab recognizes an epitope present in FS315 but not FS288.
Immunolocalization of FS proteins to prostate tumor cell
lines
To localize FS proteins, PC3 and LNCaP cells were seeded at a
concentration of 2500 cells/well on 96 well plates
(Falcon) and cultured in DMEM + 5% charcoal-stripped FCS
for 3 days. The media was removed, the cells were washed with PBS (10
mM) and fixed with Bouins fixative for 15 min at room
temperature. The cells were subsequently washed twice in 70% ethanol
and stored at 4 C in 70% ethanol before imunolocalization of FS.
The cells were washed in PBS, incubated with 6% H2O2 in PBS for 30 min, and incubated with 0.2% Triton X-100 in PBS for 15 min. After blocking with CAS Blocking solution (DAKO Corp.) for 2 h, the cells were exposed to primary antibodies overnight at 4 C. H10 (FS315 antibody) was used at a concentration of 6.75 µg/ml and 17/2 (FS288 antibody) was used at a concentration of 10 µg/ml. Following washing, the cells were incubated with biotinylated horse antimouse IgG (1/150; Vector Laboratories, Inc., Burlingame, CA) for 60 min. After three washes, the cells were incubated with the Vectastain Elite ABC Kit (Vector) for 60 min, washed, and peroxide activity was detected using a liquid 3,3'-diaminobenzidine tetrahydrochloride substrate kit (Zymed Laboratories, Inc., San Francisco, CA).
Concentration-matched mouse IgG was used as negative control for the H10 and 17/2 antibodies. In addition, the H10 Ab was preabsorbed by FS315 peptide. Preabsorption was performed by incubation of H10 with 10 times excess of FS315 peptide at 4 C. The mixture was centrifuged at 12,000 rpm, and supernatant was used for immunocyto- chemistry.
Measurement of FS production by prostate tumor cell lines
LNCaP or PC3 cells were plated at 0.5 x 105
cells/well in six-well plates (Falcon) in DMEM + 5% FCS
and cells and conditioned media were collected daily for 10 days. The
production of FS by human tumor cells was quantitated using an
ultra-sensitive ELISA (21). Briefly, cells were lysed in lysis buffer
[0.01M PBS; 0.1% vol/vol Triton X-100; 0.1% wt/vol BSA
(Sigma)] then conditioned media and cell lysates were
diluted, 1/40 and 1/10, respectively, in dissociating solution [84
mmol sodium deoxycholate; 3.4% vol/vol Tween-20; 5% wt/vol BSA;
5%vol/vol mouse serum (Sigma); 0.01 M PBS].
Standard material (hrFS288) was serially diluted in dissociating
solution to give a standard range of 2.5 ng/ml–19.5 pg/ml. Duplicate
50 µl aliquots of standard or samples were added to ELISA plates
coated with 29/9 monoclonal Ab, sealed and left overnight in a sealed
humidified container at room temperature. Plates were then washed and
50 µl of alkaline phosphatase-conjugated 17/2 antibody in Tris
conjugate buffer (25 mM Tris-HCl; 0.15 M NaCl;
1% wt/vol BSA; 0.5% vol/vol Tween 20) was added to each well. After
2 h incubation, plates were washed and 50 µl alkaline
phosphatase substrate (ELISA Amplification System; Life Technologies, Inc. Ltd, Renfrewshire, UK) was added per well.
Plates were incubated for 2 h at room temperature then 50 µl of
alkaline phosphatase amplifier (from previously mentioned kit) was
added to each well. Color development was stopped by addition of 50
µl/well 0.4 M HCl and absorbances were read at 492 nm
with 630 nm reference wavelength on a Multiscan RC microplate reader
(Labsystems and Life Sciences International UK Ltd., Basingstoke, UK)
using Genesis software (Life Sciences).
Measurement of activin A production by prostate tumor cell
lines
To quantitate production of activin A by PC3 and LNCaP prostate
tumor cells, a sensitive and specific ELISA previously used to
determine total activin A in human serum and follicular fluid (24) was
utilized. Briefly, cell lysates and standard material (hr-activin A)
were diluted in PBS + 1% BSA, and unconditioned media and media
samples were diluted in DMEM + 5% FCS. Standard material was serially
diluted to give a range of 2.5–0.005 ng/ml, whereas media and lysate
test samples were used at neat or 1/10 dilutions. Samples and standard
dilutions were denatured by the addition of an equal volume (125 µl)
of 6% SDS (wt/vol) followed by heating to 90-95 C for 4 min and
allowed to cool for 20 min at room temperature. Standards and samples
were subsequently oxidized by the addition of 20 µl 30%
H2O2. 25 µl of ELISA diluent (0.1
M Tris-HCl; 5% wt/vol BSA; 5% vol/vol Triton X-100; 0.1%
wt/vol NaN3) was added to all wells of dry E4 monoclonal
antibody-coated ELISA plates. Duplicate 100 µl aliquots of standard
or test samples were added, and the plates were left overnight in a
humidified container at room temperature. Plates were then washed and
50 µl of biotinylated E4 antibody (diluted 1:1300 in ELISA diluent)
was added for 2 h before plates were washed again. Fifty
microliters alkaline phosphatase conjugated streptavidin (diluted
1/1000 in ELISA diluent) was added to all wells for 2 h, then
plates were washed and alkaline phosphatase bound to the well was
quantitated using the same detection system used for FS ELISAs (ELISA
Amplification System; Life Technologies, Inc.) as
described above. Detection limits of the assays were determined to be
approximately 10 pg/ml.
Induction of response to activin A in PC3 cells
To block or neutralize endogenous FS production, increasing
concentrations of the 17/2 antibody were added to the tumor cells
before testing the subsequent response to activin A. PC3 cells were
plated in 96-well plates at 5000 cells/well in DMEM + 5% FCS. Cells
were left for 48 h at 37 C in humidified atmosphere in 5%
CO2. After cells had attached to wells, media was changed
to fresh DMEM + 5% FCS and 17/2 antibody or activin A were added.
Increasing concentrations of 17/2 antibody or concentration matched
mouse IgG control (10, 20,40 µg/ml) were added to cells in the
presence or absence of a constant dose of activin A (40 ng/ml). After 2
days of culture, the effects of antibodies and growth factors was
determined by the addition of 3H-thymidine (0.5 µCi/ml)
for the last 20 h of culture. Cells were harvested using a
Micromate 196 Cell Harvester (Packard Instrument Co., Meriden, CT), and levels of 3H-thymidine
incorporated were determined.
Western blotting
To detect FS proteins by Western blot, cell-conditioned media
were collected from LNCaP and PC3 prostate tumor cells, and media were
freeze dried. Media samples were reconstituted in 0.1 M PBS
and run on a 12.5% polyacrylamide nonreducing gel and separated
proteins were transferred to a 0.22 µm pore size Optitran BA-S
reinforced nitrocellulose transfer membrane (Schleicher & Schuell, Inc., Dassell, Germany) at 30 V overnight in transfer
buffer containing 20 mM Tris base, 150 mM
glycine, 20% vol/vol methanol. After transfer, membrane was stained
with Ponceau S stain (Sigma) to identify molecular weight
marker proteins, and was blocked for 1 h with 5% powdered milk in
0.01 M PBS. The membrane was then incubated with primary
antibodies for FS288 (17/2) or FS315 (H10) at a concentration of 1
µg/ml in 1% milk/0.01 M PBS/0.1% Tween 20 for 2 h,
then washed 5 x 3 min in 1% milk/PBS/Tween. The second antibody
used was a horseradish peroxidase conjugated goat antimouse IgG
(DAKO Corp.) at a dilution of 1/10000 in 1%
milk/PBS/Tween for 1 h followed by 5 x 3 min washes as
described above. Solution A and solution B of the ECL+Plus Western
blotting detection system (Amersham Pharmacia Biotech UK
Limited, Little Chalfont, Buckinghamshire, UK) were warmed to room
temperature then mixed at a ration of 40:1 and added to the membrane.
Membrane was then exposed to X-OMAT x-ray film (Eastman Kodak Co., Rochester, NY) and signal was analyzed after 2.5 min.
Data analysis
Each experiment was repeated at least three times. Where
statistical analysis was undertaken, variation was determined by ANOVA
and differences were tested using Student’s t test:
P < 0.05 (two tailed) was considered significant.
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) although FS288 levels
remained approximately 2- to 3-fold greater than activin A.
In contrast to the stable levels of activin A and FS288 observed in
cell lysates, measurement of daily production of activin A and FS288 in
PC3 cell conditioned media (Fig. 1C) revealed that activin A levels in
media were initially high but gradually decreased until a significant
reduction in production was observed after 5 days in culture
(P < 0.05, compared with Day 1); thereafter, levels of
daily activin A production remained constant. The daily levels of FS288
in conditioned media were initially low but increased steadily,
reaching a significantly higher level (P < 0.05) after
6 days, which plateaued and remained approximately 2-fold higher than
that seen over the first 5 days in culture (Fig. 1C). In PC3 cell
conditioned media, daily FS288 production was inversely correlated to
daily activin A production (r = -0.7719, P =
0.008). Overall, it can be seen that production of activin A and FS288
by PC3 cells continued over the entire culture period, although the
rate of activin production decreased with time in culture, whereas rate
of production of FS288 increased, especially over the last 5 days in
culture (Fig. 1D).
Immunoreactive FS protein was detected in LNCaP and PC3 tumor cells
using the antibody to FS315, H10 (Fig. 2A, B). No positive staining was recorded
if the antibody was preabsorbed with FS315 peptide or if concentration
matched mouse IgG was used (data not shown). In contrast,
immunoreactivity for FS288 (using the 17/2 antibody) was only detected
in the PC3 cell line and not in the LNCaP cells (Fig. 2, C and D). To
confirm the presence of FS288 and FS315 immunoreactivity in the cells,
Western blot analysis of PC3 and LNCaP media was performed. As shown in
Fig. 3A, using 17/2 antibody, two
isoforms of FS288 were present in media from PC3 cells but were not
detected in media from LNCaP cells or in hrFS315. Using the H10
antibody, two to three isoforms of FS315 protein were detected by
Western blot (Fig. 3B); no corresponding bands were detected in media
from LNCaP or PC3 and nothing was detected in hrFS288 protein.
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). However, activin A together
with increasing concentrations of the FS 288 antibody 17/2, resulted in
a dose-dependent decrease in cell proliferation, to approximately 60%
of control values (Fig. 4).
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The expression of mRNA for the activin ßA subunit is known to be present in both cell lines, and immunoreactivity for the ßA subunit has been recorded in LNCaP and PC3 cells (16, 17). Using a specific ELISA, we have been able to measure activin A production in PC3 cells but not in LNCaP cells; this is the first measurement of the ligand, activin A, in human prostate tumor cell lines. The endogenous production of measurable levels of activin A may be one reason why PC3 cells do not respond to exogenously added activin A, whereas growth of LNCaP cells (which do not produce measurable levels of activin A), is inhibited by nanogram amounts of exogenously added activin A. Whether or not activin A is up-regulated in prostate cancer needs to be determined. Anderson et al. (25) did not detect inhibin ßA subunit or activin A protein in prostate tissue from normal men who had undergone vasectomy. In contrast, we have previously reported activin ßA subunit mRNA and activin proteins in nonmalignant human prostate and in cancer. The detection of activin A in PC3 cells may suggest that the expression of the ligand is up-regulated in tumor cells; however, this is not a constant observation as LNCaP cells contained no measurable activin A.
The activin subunit ßB is also expressed by the tumor cell lines (16, 17), and it is possible that the other forms of activin, i.e. activin AB and activin B, may be synthesized by the cells. This remains to be determined, but attempts to measure the other activin ligands in this laboratory have not been successful to date; it is possible that the pattern or relative levels of activin A, B, and AB synthesis is different in the two cell lines. We have shown that both activin B, as well as activin A, inhibits cell proliferation in LNCaP cells, although those studies were limited due to the amounts of activin B that were available (14). Whatever form of activin is synthesized by the cells, the synthesis of activin A by PC3 cells is significantly higher than that from LNCaP cells, but it is unlikely to be the main reason for the lack of response by PC3 cells to activin A. A similar finding by DiSimone et al. (26) reported that endogenous production of activin A or B did not correlate with differences in proliferative activity observed in human ovarian cancer cell lines. Further work is required to measure the synthesis of other activin ligands, involving the ßA subunit, to determine if such a correlation does exist.
Alternatively, the production of FS288 by PC3 cells could contribute to the resistance to activin A. We have shown that mRNA for FS315 is expressed in LNCaP and PC3 cells, but mRNA for FS288 can be detected only in the PC3 cell line (14). This study reports that FS 288 protein is present in media from PC3, and not LNCaP cell cultures and that FS288 protein can be measured by a specific ultrasensitive ELISA. The levels of FS288 that are measured, are readily detected by the assay and estimated to be between 3 and 8 ng/ml/day in wells plated with 50,000 PC3 tumor cells. During the period of culture the pattern of FS288 and activin A synthesis is altered so that activin A levels plateau but FS288 synthesis continues to increase. The reason why FS288 synthesis increases without a corresponding change in activin A is unknown and may be related to a change in the type of activin dimers produced by the PC3 cells over the course of the culture period. However, the relative proportion of FS bound to activin A is not known because the FS288 ELISA measures "total" FS (i.e. FS previously bound to activin and free FS) and the activin A ELISA measures "total" activin A. Regardless of what proportion of FS288 is bound to activin A, the addition of FS288 antibody (17/2) to the PC3 cells, permits or induces a response to activin A. The addition of 17/2 antibody alone reduced DNA synthesis, but this lowered response was not significant. In theory it might be predicted that endogenous activin A, in the presence of the 17/2 antibody, may significantly reduce DNA synthesis. However, any growth inhibition is contingent upon exposure of the cells to an appropriate concentration of endogenous activin A. We estimated that the levels of activin A produced by the PC3 cells was less than 4 ng/ml, and this concentration may not be sufficient to cause a significant fall in 3H-thymidine incorporation: note that activin A was added at 40 ng/ml in the presence of the anti-FS288 antibody 17/2, and this dose reduced PC3 cell proliferation by approximately 50%. Thus, a lower dose of 4 ng/ml of activin A, from an endogenous (or exogenous) source, may not cause a significant response, although it may be enough to have a minor effect on DNA synthesis. This lends further support to the idea that the synthesis of FS288 by PC3 cells renders them resistant to the inhibitory effects of activin A.
Both cell lines express mRNA and protein for FS315; however, measurement of this isoform of FS remains to be determined, and accurate measurement awaits the development of a specific ELISA for FS315. Nevertheless, it is the synthesis of FS288, specifically produced by PC3 and not LNCaP cells, which may be particularly important in terms of resistance to activin A. FS288 and FS315 bind activin A with similar affinities, but FS288, rather than FS315, may play a role in the inactivation and clearance of activin A (9). FS288 exhibits a high affinity for heparan sulfate proteoglycans (HSPGs), whereas FS315 has a lower affinity. In pituitary cells, cell-associated FS288 accelerates endocytic internalization of activin A and ultimately leads to degradation by lysosomal enzymes. Hashimoto and colleagues (9) concluded that FS288 associated with cell surface HSPGs causes irreversible degradation of activin A, whereas activin A bound to FS315, is not fated for endocytic breakdown and is generally considered to be the form of FS in the systemic circulation (5, 27).
It is puzzling that PC3 prostate tumor cells would permit the synthesis of activin A, which has the potential to be growth inhibitory and induce apoptosis. A similar paradox exists in tumor tissues from men with high grade prostate cancer (13), i.e. ßA and ßB subunit mRNA expression and proteins have been localized to the tumor cells, and yet it is assumed that the tumors are actively undergoing cell proliferation and may have caused metastasis. One answer to this paradox may lie in the fact that activins have a widespread tissue distribution, and their actions are not limited to the regulation of FSH. For example, activins have roles in development, wound healing, tissue repair, cardiovascular function, and immune response (28, 29). Some of these effects may be advantageous to progression of the tumor, e.g. in the promotion of angiogenesis and immune suppression. Such actions may be achieved through circulating forms of activins bound to FS315 at other sites in the body. The local, potentially deleterious effects of activin A, could be neutralized by binding to FS288 associated with cell surface HSPGs, and the ligand degraded. Such a process would prevent/block the autocrine effect of activin A and permit tumor cell growth. In biopsy material from men with high grade cancer, we have reported the localization of FS288 to the tumor cells, in conjunction with the expression of activin ßA and ßB subunit proteins. By analogy, we may speculate that the autocrine effects of activin A on the tumor cells could be blocked by cell-associated FS288.
The tumor cell lines used in this study were originally derived from prostate metastases (30, 31), and further work is required to determine if there is any difference in the expression of FS288 in primary prostate tumors or in the sites of metastasis. Specifically, it would be important to determine which tumors do not express FS288 and if these are inhibited by activin A or other activin ligands. If this were the case, then it may be possible to develop the activin ligands as therapeutic agents for androgen-dependent and independent prostate cancer in combination with systems of targeted, specific delivery of activin to the tumor cells.
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Acknowledgments |
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Received February 23, 1999.
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) and FS288 (
)
(shown as ng protein/106 cells/day) in PC3 cell lysates
collected at 24 h intervals over 10 days in culture. Values represent mean ±
SD of five replicate wells. Figure 1C
