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Mutually Antagonistic Effects of Androgen and Activin in the Regulation of Prostate Cancer Cell Growth

Pediatric Surgical Research Laboratories, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Shyamala Maheswaran, Pediatric Surgical Research Laboratories, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Activin, a member of the TGFß superfamily, is expressed in the prostate and inhibits growth. We demonstrate that the effects of activin and androgen on regulation of prostate cancer cell growth are mutually antagonistic. In the absence of androgen, activin induced apoptosis in the androgen-dependent human prostate cancer cell line LNCaP, an effect suppressed by androgen administration. Although activin by itself did not alter the cell cycle distribution, it potently suppressed androgen- induced progression of cells into S-phase of the cell cycle and thus inhibited androgen-stimulated growth of LNCaP cells. Expression changes in cell cycle regulatory proteins such as Rb, E2F-1, and p27 demonstrated a strong correlation with the mutually antagonistic growth regulatory effects of activin and androgen. The inhibitory effect of activin on growth was independent of serine, serine, valine, serine motif phosphorylation of Smad3. Despite their antagonistic effect on growth, activin and androgen costimulated the expression of prostate-specific antigen through a Smad3-mediated mechanism. These observations indicate the existence of a complex cross talk between activin and androgen signaling in regulation of gene expression and growth of the prostate.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
ACTIVINS THAT BELONG to the TGFß class of molecules are composed of two ß subunits, ßA and ßB, which form activin A (ßAßA), activin B (ßBßB), and activin AB (ßAßB) (1). They are synthesized by many tissues and are para/autocrine regulators of cellular function (2). Follistatin, an extracellular protein that specifically binds and neutralizes activin is often coexpressed with activin and modulates its activity (3). Activin signaling in cells is transmitted by binding to a family of cell surface receptor serine threonine kinases, which include ActRIIA (activin type II receptor A) and ActRIIB (activin type II receptor B), of the type II receptor family, and ALK2 (activin-like kinase2; ActRIA) and ALK4 (ActRIB) of the type I receptor subclass (4, 5, 6). Activation of activin receptors leads to phosphorylation of Smad2 and Smad3 on two serine residues in a Ser-Ser-X-Ser motif at the end of the C-terminal domain (7, 8), complex formation of phospho-Smad2 or 3 with Smad4, and translocation to the nucleus where they modulate transcription (9). Although the most recognized role for activins is in the regulation of gonadal function (10, 11), they have also been implicated in the control of many other cellular processes including growth and tumorigenesis (12, 13, 14).

Activin and activin receptors are expressed in the epithelial cells of rat prostate (15, 16, 17) and in human benign prostatic hyperplasia and prostate cancer tissues and cell lines (18, 19, 20, 21, 22, 23, 24), suggesting that activin-mediated signaling may regulate prostate growth and development. A functional role for activin in the prostate was demonstrated by its ability to inhibit [³H] thymidine incorporation in the androgen-dependent human prostate cancer cell line LNCaP in the absence or presence of dihydrotestosterone, and in the androgen-independent prostate cancer cell line DU-145, which was reversed by the activin antagonist follistatin (20, 25). In agreement with these observations, activin treatment of LNCaP cell cultures led to a decrease in cell number (19, 26). Growth and neoplastic transformation of the prostate are finely controlled by a balance between the stimulatory effects of factors such as androgen and the opposing inhibitory effects of peptide factors including members of the TGFß family (27). Although the growth-inhibitory effects of activin on prostate cancer cells have been known for many years, the functional interaction between androgen and activin on processes such as cell cycle progression and apoptosis have never been evaluated. Such studies will provide insight into the interplay between molecules that govern growth, differentiation, and tumorigenesis of the prostate.

In addition to inhibiting prostate cancer cell growth, activin has been shown to induce the expression of prostate-specific antigen (PSA), prostatic acid phosphatase, and the androgen receptor (26), genes that are also stimulated by androgen. The molecular mechanism through which activin regulates gene expression and growth in the prostate is not known, and Smad proteins that are mediators of activin signaling (6) and coregulators of androgen receptor activity (28, 29, 30, 31), may play a role in this process.

In this manuscript, we demonstrate that activin and androgen induce the expression of PSA through a Smad3-mediated mechanism, an effect that was additive when these agents were used in combination. Despite their costimulatory effect on PSA expression, activin and androgen were mutually antagonistic in regulating prostate cancer cell growth through a Smad3-independent mechanism. Activin potently inhibited the growth of LNCaP cells maintained in androgen-depleted medium by inducing apoptosis, which was suppressed by simultaneous addition of androgen. Although activin alone did not alter the cell cycle distribution of LNCaP cultures, it suppressed androgen-induced progression of cells into the S-phase, suggesting that activin blocks androgen-stimulated growth of prostate cancer cells primarily by preventing androgen-induced cell cycle progression. These observations indicate the existence of a complex cross talk between activin and androgen signaling in the regulation of gene expression and growth of the prostate.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Dominant Negative Smad3 (3SA) Expression Blocks Activin A-Induced Smad3 Phosphorylation
LNCaP cells were stably transfected with a FLAG-tagged 3SA cDNA construct in which the three serine residues at the C-terminal serine, serine, valine, serine motif were mutated to alanine residues. The construct was sequenced to confirm that it contained the AAVA sequence described for Smad 3 (3SA) mutants (data not shown). To affirm further that the Smad3 (3SA) construct was functional as a dominant negative, its ability to inhibit TGFß-mediated induction of the 3TP-lux reporter construct in HaCaT cells was evaluated (Fig. 1A). Expression of Smad3 (3SA) abrogated the induction of the 3TP-lux luciferase construct by TGFß, suggesting that the dominant negative Smad3 construct used in subsequent experiments was indeed functional.

View larger version (28K): [in this window] [in a new window]   Fig. 1. Activin-Induced Smad3 Phosphorylation in LNCaP Cells Is Abrogated by 3SA Expression A, Dominant negative Smad3 abolishes induction of 3TP-lux-luciferase by TGFß. HaCaT cells were transfected with 0.5 µg of 3TP-lux luciferase reporter construct and either (0.5 µg) vector or (0.5 µg) FLAG-tagged Smad3 (3SA) construct. A renilla luciferase reporter construct (0.01 µg) was used to control for transfection efficiency. Cells were untreated or treated with 100 pM TGFß for 24 h and protein lysates were assayed for luciferase activity (n = 3). B, LNCaP cells were stably transfected with either 12 µg of vector or Smad3 (3SA) expression constructs along with 1 µg of hygromycin-resistance plasmid. Total protein (100 µg) isolated from two hygromycin-resistant clones was analyzed by Western blot analysis using an anti-FLAG antibody. Vector-transfected cells were used as controls. Position of the Smad3 (3SA) protein is shown. Hybridization to actin is shown to control for loading. C, Activin induces Smad3 phosphorylation in LNCaP cells. Upper panel, LNCaP cells were treated with 2 nM activin for indicated times and total protein lysates were analyzed with an anti-phospho-Smad2/3 antibody. Position of phospho-Smad3 is indicated. Lower panel, The blot was stripped and probed with an antibody specific for Smad3 protein. Position of Smad3 is shown. D, Smad3 (3SA) abrogates activin-induced phosphorylation of endogenous Smad3 protein in LNCaP cells. Upper panel, Vector and Smad3 (3SA) expressing LNCaP cells were treated with 2 nM activin for indicated times and total protein lysates were analyzed with an anti-phospho-Smad2/3 antibody. Lower panel, The blot was stripped and probed with an antibody specific for Smad3 protein. E, Protein lysates from above experiment were analyzed by Western blot using an antibody specific for phospho-Smad2. Hybridization to total Smad2 protein is shown below.

Activin A Induces PSA Expression through a Smad3-Mediated Mechanism
In agreement with previously published reports (26), activin A induced the expression of PSA in LNCaP cells (Fig. 2A). The induction of PSA in cells treated with both 5-dihydrotestosterone and activin A was additive compared with that observed with either agent alone. Similar results were observed with R1881 and activin A (data not shown).

View larger version (49K): [in this window] [in a new window]   Fig. 2. Smad3DN Reverses Activin and Androgen-Stimulated PSA Expression A, LNCaP cells maintained in androgen-depleted medium for 4 d were either untreated or treated with 2 nM activin, 10 nM 5-dihydrotestosterone (DHT), or both for 24 h. Total RNA (7.5 µg) was analyzed by Northern blot to detect PSA expression. Hybridization to 18S is shown to control for loading. B, Smad3 (3SA) suppresses activin-induced PSA expression. Vector and Smad3 (3SA) expressing LNCaP cells grown in androgen-depleted medium for 4 d were treated with 2 nM activin for 24 h. Total RNA (7.5 µg) was analyzed by Northern blot for PSA expression. Hybridization to 18S is shown to control for loading. C, Smad3 (3SA) suppresses R1881- induced PSA expression. Vector and Smad3 (3SA) expressing LNCaP cells grown in androgen-depleted medium for 4 d were treated with 10 nM R1881 for 24 h. Total RNA (7.5 µg) was analyzed by Northern blot for PSA expression. Hybridization to 18S is shown to control for loading.

Inhibition of Prostate Cancer Cell Growth by Activin A Is Independent of Smad3 Activation
In agreement with published reports (20, 25), activin inhibited the growth of LNCaP cells; addition of 2 nM activin A to cells grown in medium containing 10% serum once at the beginning of the experiment prevented growth for as long as 10 d (Fig. 3A, upper panel; P < 0.0001 between untreated and activin-treated samples on d 7 and 10). The two LNCaP cell clones stably expressing Smad3 (3SA) were simultaneously treated with activin A. As seen with parental LNCaP cells, activin prevented the growth of Smad3DN1 and Smad3DN2 cells, suggesting that overexpression of Smad3 (3SA) does not overcome the inhibitory effect of activin on prostate cancer cell growth (Fig. 3A, lower panels; P < 0.0001 between untreated and activin-treated samples on d 7 and 10).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Expression of Smad3 (3SA) Protein Does Not Abrogate Activin-Mediated Inhibition of Growth A, LNCaP cells grown in 10% fetal bovine serum containing medium were treated with 2 nM activin. Cell viability was determined after 2, 5, 7, and 10 d by MTT conversion. Plates were analyzed in an ELISA plate reader at 550 nm with a reference wavelength of 630 nm (n = 8). Statistical analysis was done using Student’s t test for paired data. P < 0.0001 between untreated vs. activin-treated samples on d 7 and 10. B, LNCaP cells grown in androgen-depleted medium for 4 d were treated with 2 nM activin, 0.1 nM R1881, or both for 7 or 10 d. Cell viability was determined by MTT conversion. Plates were analyzed as described above (n = 8). Statistical analysis was done using Student’s t test for paired data. *, P < 0.0001 between indicated samples. Vector-transfected cells are shown in the upper panels and Smad3 (3SA)-transfected cells are shown in the lower panels. Data shown are representative of three independent experiments.

Activin A was also a potent inhibitor of androgen-stimulated growth. Treatment with 100 pM R1881 led to a 2.5-fold increase in cell number on d 10, and this effect that was completely blocked in cultures treated with a combination of R1881 and activin A (P < 0.0001) and similarly activin A effectively blocked R1881-stimluated growth of Smad3 (3SA)-expressing LNCaP cells (P < 0.0001, Fig. 3B, upper and lower panels). These experiments demonstrate that inhibition of prostate cancer cell growth by activin A in the absence or presence of androgen does not require Smad3 phosphorylation.

Androgen Suppresses Activin-Induced Apoptosis and Activin Inhibits Androgen-Induced Cell Cycle Progression
Cell cycle analysis and apoptosis assays were performed to determine the mechanisms by which activin A inhibits growth. LNCaP cells grown for 4 d in androgen-depleted medium were treated with 100 pM R1881, 100 pM R1881 + 2 nM activin A, or 2 nM activin A alone for 60 h and total protein was analyzed for caspase-3 cleavage, a marker of apoptosis. Caspase-3 belongs to a family of highly conserved cysteine proteases that mediate the course of apoptotic cell suicide. Activin A strongly induced the cleavage of pro-caspase 3 to its active forms in LNCaP cells maintained in androgen-depleted medium (Fig. 4A). Another marker of apoptosis is the translocation of annexinV from the inner surface of the plasma membrane to the outside that occurs after initiation of apoptosis. LNCaP cells were treated with activin, R1881, or both for 96 h and cell surface expression of annexinV was analyzed by staining with a fluorescein isothiocyanate (FITC)-annexinV antibody. LNCaP cell cultures treated with activin demonstrated a higher degree of apoptosis compared with untreated controls (P < 0.01), and the presence of R1881 suppressed activin-induced apoptosis (P = 0.01, Fig. 4B). The potential of androgen to overcome activin-mediated apoptosis initiated under androgen-deprived conditions suggests that activin utilizes an alternate mechanism besides the induction of apoptosis to inhibit androgen-stimulated growth.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Androgen Suppresses Activin-Induced Apoptosis and Activin Inhibits Androgen-Induced Cell Cycle Progression A, LNCaP cells grown in androgen-depleted medium for 4 d were treated with 2 nM activin, 0.1 nM R1881 or both for 60 h. Total protein lysates (100 µg) were analyzed by Western blot for caspase 3 cleavage. Position of the activated p17 subunit of caspase 3 is shown. Hybridization to E-cadherin is shown to control for loading. B, LNCaP cells grown in androgen-depleted medium for 4 d were treated with 2 nM activin, 0.1 nM R1881 or both for 3 d. Cells were stained with annexin V-FITC and 4',6-diamidino-2-phenylindole and analyzed by FACS. Fold induction in apoptosis representative of the average of three independent experiments is shown. Apoptosis in untreated cells was set at 1. Statistical analysis was done using Student’s t test. C, LNCaP cells grown in androgen-depleted medium for 4 d were treated with 2 nM activin, 0.1 nM R1881, or both for 3 d. Cells were fixed in ethanol and incubated with propidium iodide and ribonuclease A. DNA content was analyzed by FACS. Analysis of the percentage of cells in the S (left panel) and G1 phase (right panel) of the cells cycle are shown. Analysis of untreated cells grown for 72 h is shown as control (n = 3). Statistical analysis was done using Student’s t test.

Inhibition of Androgen-Induced Cell Cycle Progression by Activin A Correlates with Expression of Cell Cycle/Growth Regulatory Proteins
We next analyzed whether the mutually antagonistic effects of activin and androgen on prostate cancer cell growth would correlate with the regulation of cell cycle/growth regulatory protein expression by these two ligands. In LNCaP cells, androgen-responsive growth signals are mediated through regulation of cell cycle regulatory proteins controlling the G1-S cell cycle transition. Cell cycle progression through G1 is regulated by the activity of two classes of cyclin-dependent kinase inhibitors, the CIP (p21, p27, and p57) and INK4 (p15, p16, p18, and p19) families. The cyclin-dependent kinase inhibitors inhibit the phosphorylation and activation of pocket proteins including Rb by the cyclin/cyclin-dependent kinase complexes (32). The hypo-phosphorylated pocket proteins sequester the E2F family of transcription factors, which play a role in cell the cycle progression as well as apoptosis.

LNCaP cells grown in medium containing 10% fetal bovine serum were treated with 2 nM activin for 24 h, and total protein was analyzed for the expression of cell cycle regulatory proteins. A single Immobilon-P membrane containing the proteins was cut horizontally into three segments, and the region above the 97-kDa marker was probed with anti-RB, the region below the 66-kDa marker with anti-E2F-1 and the region below 45 kDa with anti-p27 antibodies. Conforming to its ability to inhibit the growth of prostate cancer cells, activin induced hypophosphorylation of the Rb protein (Fig. 5A). Similar results were observed in LNCaP cells expressing Smad3 (3SA) (Fig. 5A), confirming the growth inhibition studies that demonstrated that phosphorylation of Smad3 may not be required for activin-mediated inhibition of LNCaP cell growth. This blot was reanalyzed to determine the effect of activin on the expression of p130, another member of the pocket protein family. As shown in Fig. 5A, activin treatment did not affect p130 expression in LNCaP cells and thus served as a control for equal loading of protein.

View larger version (54K): [in this window] [in a new window]   Fig. 5. Regulation of Cell Cycle Regulatory Protein Expression by Activin A, Vector and Smad3DN expressing LNCaP cells grown in 10% fetal bovine serum containing medium were treated with 2 nM activin for 24 h. Total protein (100 µg) was analyzed by Western blot for expression of the Rb protein. Lower panel, The blot from above was stripped and reprobed with an anti-p130 antibody. Positions of hypophosphorylated (Rb) and hyperphosphorylated (P-Rb) Rb proteins and p130 are shown. B, The immunoblot from above was probed with an anti-E2F-1 antibody. Position of the E2F-1 protein is shown. C, The immunoblot from above was probed with an anti-p27 antibody. Position of the p27 protein is shown.

Activin-mediated changes in p27 expression was not evident in LNCaP cells grown in medium containing 10% fetal bovine serum (Fig. 5C; see Fig. 6C).

View larger version (38K): [in this window] [in a new window]   Fig. 6. Regulation of Cell Cycle/Growth Regulatory Protein Expression by Activin and Androgen Correlates with their Mutually Antagonistic Effects on Prostate Cancer Cell Growth A, LNCaP cells grown in androgen-depleted medium for 4 d were treated with 2 nM activin, 0.1 nM R1881, or both for 24 h. Total protein lysates (100 µg) were analyzed by Western blot for expression of the Rb protein. Lower panel, The blot was stripped and reprobed with an anti-p130 antibody. Positions of hypophosphorylated (Rb) and hyperphosphorylated (P-Rb) Rb proteins and p130 are shown. B, The immunoblot from above was probed with an anti-E2F-1 antibody. Position of the E2F-1 protein is shown. C, The immunoblot from above was probed with an anti-p27 antibody. Position of the p27 protein is shown.

In concordance with its ability to inhibit androgen-mediated entry of LNCaP cells into S-phase of the cell cycle, activin reversed R1881-induced hyperphosphorylation of the Rb protein (Fig. 6A) but did not affect Rb protein phosphorylation when used alone. Reanalysis of this blot with an anti-p130 antibody demonstrated no difference in p130 expression between the samples and also served as a control for equal loading of protein (Fig. 6A).

Hyperphosphorylation of Rb protein during progression of cells from the G1 phase of the cell cycle to the S-phase leads to the release of E2F-1, which in turn stimulates its own expression. Consistent with the hyperphosphorylation of Rb, E2F-1 protein level increased in R1881-treated LNCaP cells, an effect reversed by simultaneous addition of activin. Moreover, activin itself strongly suppressed E2F-1 protein levels in LNCaP cells (Fig. 6B).

Interestingly, the mutually antagonistic effect of activin and androgen was also evident in the regulation of cellular p27. Activin treatment of androgen-deprived LNCaP cells induced the expression of p27 and cotreatment with androgen suppressed this effect (Fig. 6C). The increase in p27 after activin treatment may not have been evident in LNCaP cells grown in medium containing 10% fetal bovine serum (Fig. 5C), most likely due to the suppressive effect of androgens in the medium.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Activin plays a critical role in the tumorigenesis of a variety of tissues (33, 34, 35). The presence of activins and its receptors in the prostate (15, 16, 17, 18, 19, 20, 21, 22, 23, 24) and the ability of activin A to inhibit prostate cancer cells grown in culture (19, 20, 25, 26) suggest a role for activins in the regulation of prostatic growth. Activin has been shown to induce apoptosis in many cell lines (36, 37, 38, 39, 40), and Smad proteins have been shown to play a role in this process. In HS-72 B cell hybridoma, activin-mediated DNA fragmentation correlated with phosphorylation of Smad2 whereas constitutive expression of Smad7, an antagonistic Smad protein, abrogated Smad2 phosphorylation and DNA fragmentation (41), suggesting that receptor-mediated phosphorylation of Smad proteins may be required for induction of apoptosis. Although other studies have also implicated Smad2 in activin-induced apoptosis (39, 40), the contribution of Smad3 phosphorylation on the carboxy-terminal serine, serine, valine, serine motif to activin-stimulated gene expression, apoptosis, and cell cycle inhibition is not clear. Our studies show that phosphorylation of Smad3 is essential for stimulation of PSA expression by activin but is not required for its growth inhibitory effect in prostate cancer cells. The early diagnosis of prostate cancer has been facilitated by the development of serum PSA testing. The induction of PSA by factors such as activin that possess growth inhibitory activity may contribute to the lack of specificity of PSA in prostate cancer screening.

Activin inhibits cell cycle progression in many cell types through regulation of cell cycle regulatory molecules and hypophosphorylation of the Rb protein (38, 41, 42, 43, 44). Activin’s reversal of androgen-induced Rb phosphorylation and E2F-1 tightly correlated with its ability to reverse R1881-induced entry of cells into S-phase of the cell cycle. In addition, activin alone strongly suppressed E2F-1 in LNCaP cells grown in androgen-depleted medium. Park et al. (45) recently demonstrated that the lowering of E2F-1 protein levels by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin was a critical step in the induction of apoptosis in prostate cancer cells and that the overexpression of E2F-1 suppressed lovastatin-stimulated apoptosis and promoted cell survival. The pattern of E2F-1 regulation by both activin and androgen suggests that it may be one of the factors responsible for their mutually antagonistic effect on growth.

Interestingly, activin also increased p27 protein expression in LNCaP cells grown in androgen-depleted medium, an effect suppressed by the presence of androgen. Although exogenous p27 expression in LNCaP cells results in both induction of apoptosis and cell cycle regulation (46), the lack of difference in p27 between R1881 and R1881 + activin-treated LNCaP cells indicates that p27 is unlikely to play a role in activin-mediated reversal of androgen-induced entry into S-phase of the cell cycle. However, the induction of p27 by activin and its suppression by simultaneous addition of R1881 correlates with the ability to antagonize activin-induced apoptosis, suggesting that it may be an intermediary signaling molecule in this process.

Our observations indicate that the growth inhibitory effect of activin A in LNCaP cells in the absence of androgen occurs primarily through induction of apoptosis, which is completely blocked in the presence of androgen. Although activin alone did not interfere with cell cycle progression, it inhibited R1881-stimulated entry of cells into S-phase of the cell cycle (Fig. 7). The antagonistic interaction between androgen and activin in the regulation of proliferation and apoptosis may be relevant in androgen-dependent processes such as branching morphogenesis, castration-induced regression, and neoplastic transformation of the prostate. Testosterone administration to prostatic explants induces proliferation and extensive branching of the prostatic epithelial ducts, and administration of activin A inhibited this process. In agreement with the ability of activin to reverse androgen-induced cell cycle progression reported in here, inhibition of this testosterone-induced branching process by activin was associated with reduced proliferation and was not due to increased apoptosis in any compartment of the prostate explants (17).

View larger version (23K): [in this window] [in a new window]   Fig. 7. A Model that Depicts the Cross Talk between Activin and Androgen in Prostate Cancer Cells Activin and androgen can costimulate PSA expression through a Smad3-dependent mechanism. Activin blocks androgen-independent survival of prostate cancer cells by inducing caspase3 cleavage and apoptosis. Androgen inhibits these activin-induced events and stimulates S-phase entry, which leads to the growth of prostate cancer cells. Activin in turn antagonizes androgen-stimulated cell cycle progression and ultimately inhibits androgen-induced growth.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Cells, Reagents, and Growth Assays
The human prostate cancer cell line LNCaP was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, glutamine, and penicillin/ streptomycin. The Smad3 (3SA) construct was a kind gift from Dr. Patricia Donahoe’s laboratory at Massachusetts General Hospital. LNCaP cells stably expressing the dominant negative Smad3 protein were generated by transfecting the cells with 1 µg of hygromycin resistance plasmid and 12 µg of Smad3DN using Fugene-6. Cells were grown in medium containing 100 µg/ml of hygromycin, and Smad3DN expressing clones were identified by Northern and Western blot analysis.

To measure the effect of activin A on LNCaP cell growth, cell suspensions (2000 cells/well) in RPMI containing 10% serum were transferred to a 96-well microtiter plate. Cells were either untreated or treated with 2 nM activin A. To measure the effect of activin on androgen-induced LNCaP growth, cells were seeded in a 96-well plate in RPMI containing 7% charcoal-stripped serum (Gemini Bio-Products Inc., Calabasas, CA). After 4–5 d of growth in androgen-depleted medium, cells were treated with the androgen analog R1881 (methyltrienolone, Perkin-Elmer, Boston, MA), activin A or R1881 + activin A. R1881 was added to the cells at a concentration of 100 pM. Recombinant human activin A was purchased from R & D Systems, Inc., Minneapolis, Minnesota, and added to the cells at a final concentration of 2 nM either alone or with R1881.

Growth was estimated based on the colorimetric reduction of a yellow tetrazolium salt, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], to a purple formazan by viable LNCaP cells. The number of viable cells was estimated by adding 30 µl of MTT solution (5 mg/ml in PBS). After 3–4 h of incubation at 37 C, during which time viable cells reduced the yellow MTT salt to its purple formazan, the stain was eluted into 200 µl of dimethylsulfoxide by agitating the plates for 5 min on a shaker. The ODs were quantified at a test wavelength of 550 nm and a reference wavelength of 630 nm on a multiwell spectrophotometer. Statistical significance was determined using Student’s t test.

Northern Blot Analysis
The 509-bp human PSA probe was derived by PCR from LNCaP cDNA using the following primers; sense, 5' AAG ACT CAA GCC TCC CCA GTT C 3'; antisense, 5' CTT CAA TAC ACC TCC CCC CAT AG 3'. Total RNA isolated from cells was probed with radiolabeled PSA or 18S ribosomal RNA as described (49).

Antibodies and Western Blot Analyses
The mouse monoclonal anti-Rb antibody was a kind gift from Dr. Edward Harlow (Harvard Medical School, Cambridge, MA). The rabbit anti-p130 antibody, rabbit anti-phospho-Smad2/3 (Ser 433/435) antibody, rabbit anti-Smad3 (FL-425) antibody, and the rabbit anti-E2F-1 antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-FLAG M2 monoclonal antibody and the anti-cleaved caspase-3 antibody that detects endogenous levels of activated caspase-3 resulting from cleavage (Asp175) were purchased from Sigma (St. Louis, MO) and Cell Signaling Technology, Inc. (Beverly, MA), respectively. The antibody that specifically recognized phospho-Smad2 alone (Ser 465/467) was purchased from Cell Signaling Technology, Inc. (Beverly, MA). Proteins were harvested in RIPA buffer [50 mM HEPES (pH 7.0), 150 mM NaC1, 0.1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS], fractionated on SDS-polyacrylamide gels, transferred to Immobilon-P (Millipore, Bedford, MA) membrane and Western blot analysis was done as described (50). The blot was stained with amidoblack (Sigma, St. Louis, MO) to ensure equal loading.

Cell Cycle Analyses
The cell cycle distribution of untreated and R1881 +/- activin-treated cultures was analyzed by fluorescence-activated cell sorting (FACS). Cells were detached with PBS/EDTA, fixed in 95% ethanol and treated with propidium iodide and ribonuclease A. Flow cytometric analysis was performed on a Becton Dickinson (Franklin Lakes, NJ) FACScan flow cytometer. To measure the apoptosis, cells were immunostained with a FITC-conjugated anti-annexinV antibody, counterstained with 4',6-diamidino-2-phenylindole and analyzed by FACS.

Statistical Analysis
The results presented are representative of three or more similar experiments and are represented as mean ± SD. Statistical analyses were performed using Student’s t test for paired data. Difference at P < 0.05 was considered to be significant.

	ACKNOWLEDGMENTS

 
We thank Drs. Alan Schneyer, Herb Lin, Jose Teixeira, and Chris Houk for critically reading this manuscript.

	FOOTNOTES

 
This work was supported by the Hershey Family Foundation and Survivors Walk for Prostate Cancer, the Claflin Distinguished Scholar Award, and NIH/National Cancer Institute Grant CA89138-01A1 (to S.M.).

J.L.C. and L.M.S. contributed equally to this work.

Abbreviations: ActRIIA, Activin type II receptor A; ActRIIB, activin type II receptor B; ALK2, activin-like kinase 2; ßAßA/ßBßB/ßAßB), two ß subunits, ßA and ßB that form activin A, activin B, and activin AB; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; MTT, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PSA, prostate-specific antigen; 3SA, dominant negative Smad3.

Received for publication September 16, 2003. Accepted for publication December 11, 2003.

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TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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