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Mullerian Inhibiting Substance Regulates Androgen-Induced Gene Expression and Growth in Prostate Cancer Cells through a Nuclear Factor-B-Dependent Smad-Independent Mechanism

Department of Surgical Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Dr. Shyamala Maheswaran, Jackson 904, Surgical Oncology, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: maheswaran{at}helix.mgh.harvard.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Mullerian inhibiting substance (MIS), a member of the TGFß superfamily, causes regression of the Mullerian duct in male embryos. The presence of MIS type II and type I receptors in tissues and cell lines derived from the prostate suggests that prostate is a likely target for MIS. In this report, we demonstrate that MIS inhibits androgen-stimulated growth of LNCaP cells and decreases their survival in androgen-deprived medium by preventing cell cycle progression and inducing apoptosis. Expression of dominant-negative Smad1 reversed the ability of MIS to decrease LNCaP cell survival in androgen-deprived medium but not androgen-stimulated growth, whereas abrogation of nuclear factor-B (NFB) activation ablated the suppressive effects of MIS on both androgen-stimulated growth and androgen-independent survival. The effect of MIS on androgen-induced growth was not due to changes in androgen receptor expression. However, MIS suppressed androgen-stimulated transcription of prostate-specific antigen; ablation of NFB activation reversed MIS-mediated suppression of prostate-specific antigen. These observations suggest that MIS regulates androgen-induced gene expression and growth in prostate cancer cells through a NFB-dependent but Smad1-independent mechanism. Thus, MIS, in addition to potentially regulating prostate growth indirectly by suppressing testicular testosterone synthesis, may also be a direct regulator of androgen-induced gene expression and growth in the prostate at the cellular level.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
MULLERIAN INHIBITING SUBSTANCE (MIS) is a member of the TGFß superfamily, a class of molecules that regulate growth, differentiation, and apoptosis in many cell systems. MIS, in male embryos, causes regression of the Mullerian ducts, the anlagen of fallopian tubes, uterus, and upper vagina in females (1). The MIS ligand is produced at high levels by Sertoli cells of the embryonic testis until puberty and continues to be synthesized at lower levels by the adult testis throughout life (2, 3). The MIS type II receptor, a transmembrane serine/threonine kinase homologous to other members of the TGFß family of type II receptors, is expressed at high levels in the Mullerian duct, Sertoli cells, and granulosa cells of the embryonic and adult gonads (4, 5, 6). Lower levels of expression have been demonstrated in nongonadal tissues including the prostate and the breast (7, 8, 9, 10). However, the functional significance of MIS-mediated signaling in these tissues remains to be determined.

The binding of MIS ligand to MIS type II receptor initiates a signaling cascade that is dependent on the recruitment of a type I receptor. Activin-like kinase 2 (ALK2), ALK3, and ALK6 have been implicated in mediating MIS signaling in cells (11, 12, 13, 14). MIS-induced type I and type II receptor complexes phosphorylate the receptor-activated Smad (R-Smad) proteins, specifically Smads 1, 5, and 8 (12). In the mouse Leydig cell tumor cell line, MA-10, MIS induces the expression of antagonistic Smads 6 and 7 (12), which inhibit signaling by TGFß family members by either binding to R-Smad or by blocking their access to the type I receptors (15, 16).

The existence of measurable levels of MIS in the serum of males and females even after regression and differentiation of the Mullerian duct (2, 3) implies a postnatal function for this gonadal hormone. In addition to causing apoptotic regression of the Mullerian duct in male embryos, MIS also regulates testosterone homeostasis by inhibiting the transcription of P450c17 hydroxylase/lyase mRNA, which encodes the enzyme responsible for the conversion of progesterone to androstenedione (17, 18). Thus, MIS may indirectly regulate testosterone-mediated processes including growth of the prostate, a tissue dependent on androgens for growth, differentiation, maintenance, and function.

Members of the TGFß superfamily have been implicated in regulating androgen-dependent processes such as branching morphogenesis (19, 20, 21), and neoplastic transformation and tumorigenicity in the prostate (22, 23, 24, 25). Although the prostate develops around the prostatic utricle, a vestigial Mullerian duct remnant (26), and MIS suppresses the biosynthesis of testosterone (17, 18), a key regulator of prostate growth, and MIS receptors are expressed in the prostate (9), suggesting that MIS may also act directly on the prostate, very little is known about MIS regulation of prostate growth and tumorigenesis.

We recently demonstrated that MIS strongly induced nuclear factor-B (NFB) DNA binding activity containing p65 and p50 subunits, led to the induction of the immediate-early gene, IEX-1, and inhibited the growth of LNCaP cells (7, 9). However, whether MIS inhibits prostate cancer cell growth by specifically antagonizing androgen activity and whether NFB and/or Smad1 activation may be required for this process was not known. In this report, we demonstrate that MIS reverses androgen-stimulated gene expression and growth in prostate cancer cells through a NFB-dependent but Smad1-independent mechanism.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
MIS Inhibits Androgen-Stimulated Growth and Androgen-Independent Survival of LNCaP Cells
Because members of the TGFß family have been implicated in regulating growth in the prostate, we tested the effect of MIS on LNCaP cell growth. The addition of a single dose of 35 nM MIS to LNCaP cells (grown in 10% fetal bovine serum-containing medium) inhibited growth by 50, 72, and 84% after 5, 7, and 10 d compared with untreated controls (n = 8; P < 0.0001; Fig. 1A). The concentration of MIS used in our experiments is consistent with the amount of MIS required to cause regression of the Mullerian duct in organ culture assays and with other experimental systems described (11, 27, 28). This is due to the purification process during which MIS retains its specificity but loses biological potency, due in part to aggregation as demonstrated by light scattering (MacLaughlin, D. T., and P. K. Donahoe, unpublished observations).

View larger version (26K): [in this window] [in a new window]   Fig. 1. MIS Antagonizes LNCaP Cell Growth in the Presence and Absence of Androgen A, LNCaP cells grown in 10% fetal bovine serum-containing medium were treated with 35 nM MIS once at the beginning of the experiment, and growth was monitored using MTT assays. P < 0.001 between untreated and MIS-treated samples on d 2, 5, 7, and 10. The experimental result shown in this figure is representative of more than three experiments. B, LNCaP cells grown in medium containing charcoal-stripped serum were treated with 0.1 nM R1881, MIS (35 nM), or R1881 and MIS. Growth after 3, 5, 7, and 10 d was estimated using the MTT assay (n = 8). The experimental result shown in this figure is representative of three such experiments.

We next determined whether MIS-mediated suppression of LNCaP cells growth results from induction of apoptosis and/or interference with cell cycle progression. LNCaP cells grown in androgen-deprived medium were treated with 0.1 nM R1881, 35 nM MIS, or both, and proteins were analyzed for cleavage of caspase-3, which serves as a marker of early-stage apoptosis. MIS induced caspase-3 cleavage in LNCaP cells, an effect not reversed by the presence of R1881 (Fig. 2A). Analysis of cell cycle progression using fluorescence-activated cell sorting demonstrated that MIS overcomes androgen-induced entry of cells into S phase of the cell cycle (Fig. 2B). Thus, MIS inhibits prostate cancer cell growth by preventing cell cycle progression and inducing apoptosis.

View larger version (41K): [in this window] [in a new window]   Fig. 2. MIS Inhibits the Growth of Prostate Cancer Cells by Preventing Cell Cycle Progression and Inducing Apoptosis A, LNCaP cells were grown in RPMI containing 5% charcoal-stripped serum for 3 d and treated with 0.1 nM R1881, 35 nM MIS, or both for 24 h, and proteins were analyzed by Western blot using an antibody against cleaved caspase-3. Proteins extracted from cells treated with 1 µM staurosporine for 24 h were used as positive control. Blot was stained with E-cadherin to control for loading. U, Untreated; M, MIS; A, R1881; M+A, MIS and R1881; S, staurosporine. B, LNCaP cells were grown in RPMI containing 5% charcoal-stripped serum for 3 d and treated with 0.1 nM R1881, 35 nM MIS, or both for 72 h and analyzed by fluorescence-activated cell sorter (FACS). Percentage of cells in the S phase is shown. Representative FACS histograms of fluorescence intensity vs. cell number are shown below. U, Untreated; M, MIS; A, R1881; M+A, MIS and R1881.

View larger version (35K): [in this window] [in a new window]   Fig. 3. The Role of Smad1 and NFB in MIS-Mediated Inhibition of Prostate Cancer Cell Growth A, Smad1DN expression abolishes the induction of BRE-luciferase by BMP-2. LNCaP cells were transfected with the BRE-luciferase reporter construct (0.02 µg) along with vector (0.18 µg) and Smad1DN (0.18 µg) plasmids. A Renilla luciferase reporter construct (0.006 µg) was used to control for transfection efficiency. Cells were untreated or treated with 1 ng/ml BMP-2 for 6 h, and protein lysates were assayed for luciferase activity (n = 4). The bars represent the SD from the mean. B, LNCaP cells were stably transfected with either 12 µg vector or Smad1DN expression constructs along with 1 µg hygromycin-resistance plasmid. Total RNA isolated from two hygromycin-resistant clones was analyzed by Northern blot. Position of the Smad1DN mRNA is shown. Hybridization to 18S was done to control for loading. C, Smad1DN mitigates BMP-induced phosphorylation of the Smad1 protein in LNCaP cells. Upper panel, Vector and Smad1DN-expressing LNCaP cells were treated with 6.25 ng/ml BMP-2 for 1 h, and total protein was immunoblotted with the anti-phospho-Smad1 antibody. Lower panels, The blots were stripped and probed again with antibodies specific for total Smad1 and E-cadherin to control for loading. D, Smad1DN mitigates MIS-induced phosphorylation of the Smad1 protein in LNCaP cells. Upper panel, Vector and Smad1DN-expressing LNCaP cells were treated with 35 nM MIS for 6 h, and total protein was immunoblotted with the anti-phospho-Smad1 antibody. Lower panels, The blots were stripped and probed again with antibodies specific for total Smad1 and E-cadherin to control for loading. E, Role of Smad1 in MIS-mediated inhibition of prostate cancer cell growth. LNCaP cells expressing Smad1DN were grown in medium containing 5% charcoal-stripped serum for 3 d and treated with 0.1 nM R1881, MIS (35 nM), or both. Vector-transfected LNCaP cells were used as control. Growth after 10 d was estimated using the MTT assay (n = 8). Statistical analysis was done by Student’s t test.

To characterize the importance of MIS-mediated Smad1 phosphorylation in MIS-mediated inhibition of LNCaP cell growth, vector and Smad1DN-expressing LNCaP cells were grown in androgen-depleted medium for 4 d and treated with 35 nM MIS, 0.1 nM R1881, and MIS plus R1881. R1881 induced the growth of both control and Smad1DN-expressing LNCaP cells (n = 8; P < 0.0001 between R1881-treated and untreated samples). MIS inhibited R1881-induced growth in all cell lines (n = 8; P < 0.01 between R1881-treated and R1881 plus MIS-treated samples). The responsiveness of the control and Smad1DN-expressing cells to R1881 and R1881 plus MIS was comparable. However, the ability of MIS to suppress androgen-independent survival was abrogated in LNCaP cells expressing the dominant-negative Smad1 (Fig. 3E). These results suggest that Smad1 is required for MIS-mediated inhibition of prostate cancer cells surviving in the absence of androgen, but not required for MIS-mediated inhibition of androgen-stimulated growth.

We had previously demonstrated that MIS robustly induces NFB activation in LNCaP cells (7, 9). The NFB family of transcriptional activators exists in the cytosol bound to the inhibitory IB family of molecules. Activation of the pathway by extracellular signals leads to phosphorylation and degradation of IB with subsequent nuclear localization of NFB (29, 30). To investigate the contribution of NFB activation to MIS-mediated inhibition of prostate cancer cell growth, we analyzed the MIS responsiveness of LNCaP cells, which stably express the dominant-negative inhibitory IB, IB (IBDN). In the IBDN transgene, serines 32 and 36, which represent the phosphorylation sites of wild-type IKB, are converted to alanines. Thus, it cannot be phosphorylated or targeted for degradation (31) and functions as a superrepressor of NFB activation. Characterization of LNCaP cells expressing IBDN is described (9). MIS induced NFB DNA binding in the vector-transfected LNCaP cells. Antibody supershifts demonstrated that the complex contained the p50 and p65 subunits of NFB. NFB activation by MIS was completely abrogated in cells expressing IBDN (Fig. 4A).

View larger version (39K): [in this window] [in a new window]   Fig. 4. Role of NFB in MIS-Mediated Inhibition of Prostate Cancer Cell Growth A, Control and IBDN-expressing LNCaP cells were treated with MIS for 1 h, and nuclear proteins were analyzed by gelshift assay using a radiolabeled NFB oligonucleotide as described in Ref. 7 . For supershift assays, 1 µg each of anti-p50 and anti-p65 antibodies were simultaneously added to the binding reaction. Positions of the NFB/DNA complexes (black arrowhead) and the supershifted complexes (open arrowhead) are shown. B, Abrogation of NFB activation mitigates the ability of MIS to suppress androgen-induced growth. Control and IBDN-expressing LNCaP cells were seeded in 96-well plates in medium containing charcoal-stripped serum and treated with 0.1 nM R1881, MIS (35 nM), or both. Growth after 5, and 10 d was estimated using the MTT assay (n = 8). C, The MIS-inducible immediate-early gene IEX-1 inhibits LNCaP growth. Equal number of LNCaP cells was transfected with 0.3 µg puromycin resistance plasmid and vector, MIS, and IEX-1 expression plasmids (2 µg each) as shown. Cultures were grown in 1 ng/ml puromycin-containing medium for 3 wk, and drug-resistant clones were stained with crystal violet and counted (n = 3). A representative well from vector, MIS, and IEX-1 transfected cultures is shown below. Lowest panel, To test the expression of the constructs, 1 µg each of IEX-1 and MIS expression constructs were transfected into COS7 cells, and total cellular proteins and culture supernatants, respectively, were analyzed by Western blot. Vector-transfected cells are shown as controls.

Because we had previously demonstrated that MIS-induced NFB activation leads to stimulation of the immediate-early gene IEX-1 (9), we tested whether expressing IEX-1 would influence the growth of LNCaP cells. Vector, IEX-1, and MIS-expressing plasmids were transfected in to LNCaP cells along with a plasmid encoding puromycin resistance and the inhibition of drug-resistant colony growth was monitored. As shown in Fig. 4C, both MIS and IEX-1 expression suppressed colony growth compared with that observed in vector-transfected cultures suggesting that IEX-1, a downstream target of MIS-mediated NFB activation, can suppress LNCaP cell growth.

MIS Suppresses Prostate-Specific Antigen (PSA) Expression through a Smad1-Independent NFB-Dependent Mechanism
To determine whether MIS overcomes the growth-stimulatory effects of androgens in LNCaP cells through down-regulation of the androgen receptor, cells grown in androgen-depleted medium for 4 d were treated with 0.1 nM R1881, 35 nM MIS, or a combination of R1881 and MIS for 24 h. Total cellular protein was analyzed by Western blot using an anti-androgen receptor antibody. As reported previously, R1881 up-regulated the expression of the androgen receptor (32), an androgen-responsive gene. Simultaneous addition of MIS did not affect receptor up-regulation by R1881 (Fig. 5A), suggesting that mitigation of androgen-stimulated growth by MIS is not due to a decrease in androgen receptor expression.

View larger version (30K): [in this window] [in a new window]   Fig. 5. MIS Suppresses Androgen-Induced PSA Expression A, LNCaP cells grown in androgen-depleted medium for 4 d were treated for 24 h with 0.1 nM R1881, 35 nM MIS, or both. Equal amount of total protein (100 µg) from samples was analyzed for androgen receptor expression by Western blot. Position of the androgen receptor (AR) protein is indicated. B, LNCaP cells grown in RPMI 1640 containing 10% fetal bovine serum were treated with 35 nM MIS for 24 h. Total RNA (7.5 µg) was analyzed for PSA expression by Northern blot. Hybridization to 18S is shown as loading control. C, MIS suppresses androgen-induced PSA expression. LNCaP cells grown in androgen-depleted medium for 4 d were treated with 0.1 nM R1881, 35 nM MIS, or both for 6 and 24 h, and total RNA was analyzed by Northern blot. Hybridization to 18S RNA is shown to control for loading. D, LNCaP cells grown in androgen-deprived medium for 24 h were transfected with PSA promoter-luciferase along with MIS and Smad1DN expression constructs as indicated in the figure. The total amount of DNA transfected into cells was equalized with vector DNA. A SV40-Renilla luciferase reporter construct was included to normalize for transfection efficiency. Twenty-four hours after transfection, cells were treated with 100 pM R1881 for 1 d, and luciferase activity was determined. The mean luciferase activity ± SD in each sample is shown. (P < 0.05 between R1881-treated, vector- and MIS-transfected samples by Student’s t test). The experimental result shown in this figure is representative of three such experiments. E, LNCaP cells grown in androgen-deprived medium for 24 h were transfected with PSA promoter-luciferase along with MIS and IBDN expression constructs as shown in the figure. The total amount of DNA transfected into cells was equalized with vector DNA. A SV40-Renilla luciferase reporter construct was included to normalize for transfection efficiency. Twenty-four hours after transfection, cells were treated with 0.1 or 10 nM R1881 for 24 h and luciferase activity was determined. The mean luciferase activity ± SD in each sample is shown (P < 0.05 between R1881-treated vector and MIS-transfected samples and MIS-transfected and MIS plus IBDN-transfected samples by Student’s t test). The experimental result shown in this figure is representative of two such experiments.

We then determined whether the ability of MIS to inhibit androgen-induced PSA expression occurred at the transcriptional level. A luciferase reporter construct under the control of the PSA promoter was transfected into LNCaP cells, and its activity was assayed. R1881 treatment of cells induced PSA promoter activity by approximately 3.5-fold compared with vehicle-treated samples. Cotransfection of MIS repressed the activity of the PSA promoter, suggesting that MIS represses androgen-induced PSA expression at the transcriptional level. The MIS expression construct used in this experiment produces biologically active MIS, and its expression has been shown to inhibit growth (8, 33). Expression of Smad1DN did not reverse MIS-mediated repression of PSA promoter activity, suggesting that this process was not dependent on activation of Smad1 (Fig. 5D).

Because MIS robustly activates that NFB pathway (Fig. 4A), we tested whether MIS-mediated suppression of PSA expression was dependent on this pathway. The effects of MIS on R1881-induced PSA promoter activity was measured in the presence and absence of the IBDN transgene, which suppresses MIS-induced NFB activation (31). Our results demonstrate that disabling NFB activation abrogates the ability of MIS to suppress R1881-induced PSA expression (Fig. 5E).

	DISCUSSION

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MIS regulates serum testosterone levels by inhibiting the transcription of P450c17 hydroxylase/lyase mRNA (17, 18) and thus may be an indirect regulator of androgen-dependent processes such as growth, differentiation, and function of the prostate gland. In fact, androgen-dependent expansion of the prostate gland in males occurs after puberty when MIS levels decline and testicular androgens begin to rise (34, 35). The presence of MIS type II and type I receptors in the prostate (7, 9) suggest that MIS-mediated signaling may also act on the prostate at a cellular level.

Human prostate tumors and cancer cell lines express ligands and receptors belonging to the TGFß superfamily (22, 23, 24), which play a regulatory role in development, differentiation, and neoplastic transformation of the prostate. Prostate cancer cells that acquire resistance to the apoptotic effect of TGFß, frequently have loss of TGFß receptors (25). There is also evidence that activin signaling may be impaired in prostate tumors; prostate tumors express significantly lower levels of activin receptor 1B mRNA when compared with nonmalignant tissue (24). Mitre et al. (36) recently diagnosed a 56-yr-old man with prostate cancer who also presented with persistent Mullerian duct syndrome. Although the molecular defect that leads to the retention of the Mullerian duct has not been identified in this patient, the vast majority of patients with persistent Mullerian duct syndrome demonstrate either a failure to synthesize MIS or have germline mutations in the MIS type II receptor (37, 38).

Our observations demonstrate that MIS can inhibit androgen-independent survival as well as androgen-induced growth of prostate cancer cells. Although expression of dominant-negative Smad1 abrogated the ability of MIS to suppress androgen-independent survival, it was not very effective in reversing the antagonistic effect of MIS on androgen-stimulated growth. These results suggest that other pathways besides activation of Smad1 may be involved in MIS-mediated inhibition of androgen-stimulated prostate cancer cell growth. We had previously demonstrated that MIS induces the NFB pathway and inhibits the growth of LNCaP cells (9). Characterizing the growth of LNCaP cells maintained in androgen-deprived medium demonstrated that disabling NFB activation with IBDN, a repressor of NFB activation, abrogates the ability of MIS to overcome androgen-stimulated growth.

In addition to overcoming androgen-induced growth, MIS was also able to inhibit androgen-induced expression of PSA mRNA. The effect of MIS on PSA expression was selective, because androgen receptor, another gene up-regulated in response to androgen (32), was not affected. Characterization of MIS-mediated inhibition of PSA expression demonstrated that it was independent of Smad1 activation but dependent on induction of the NFB pathway.

Activation of NFB is associated predominantly with increased cell survival, although in some experimental systems it is known to elicit the opposite effect (39). Our results show that MIS-mediated inhibition of LNCaP growth is dependent on NFB activation. In LNCaP cells, MIS, the growth inhibitor, and dihydrotestosterone (DHT), the growth inducer, both stimulate NFB activity containing p50 and p65 subunits but with different kinetics. MIS-induced NFB activation occurs early, whereas DHT-mediated induction occurs only after 24 h of exposure. However, MIS-induced NFB led to the expression of the immediate-early gene, IEX-1, whereas DHT did not (9), suggesting that NFB-regulated gene expression is dependent on the type of stimulus. Thus, the dual role of NFB in regulating cell survival is possibly due to differential regulation of gene expression in response to various stimuli.

The role the TGFß family on development, differentiation, and neoplastic transformation of the prostate is being extensively investigated (25, 40, 41, 42, 43, 44, 45). However, little is known about the role of MIS on regulation of prostate growth and function. Suppression of testosterone production and function is the goal for hormonal therapy of androgen-dependent prostate tumors. Our results for the first time demonstrate that MIS, in addition to suppressing testosterone synthesis (17, 18), may also be a negative regulator of androgen-stimulated gene expression and growth in the prostate at the cellular level. Thus, MIS could represent a novel naturally occurring hormonal adjuvant that can act at multiple levels to regulate androgen-dependent prostate cancer growth.

	MATERIALS AND METHODS

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Cells, Reagents, and Growth Assays
The human prostate cancer cell line LNCaP was grown in RPMI 1640 supplemented with 10% fetal bovine serum, glutamine, and penicillin/ streptomycin. To estimate growth, cell numbers were obtained using the colorimetric reduction of a yellow tetrazolium salt, MTT, to a purple formazan by viable LNCaP cells as described (46). Briefly, cells (2000 cells/ml) were seeded in RPMI containing 10% serum. Medium was removed after 24 h and RPMI containing 5–7% charcoal-stripped serum (Gemini Bio-Products, West Sacramento, CA) was added to the cells. Cells were maintained for 4 d in androgen-depleted medium and the androgen analog R1881 (methyltrienolone; PerkinElmer, Boston, MA), MIS, or R1881 plus MIS was added at concentrations described in Results and the figure legends. The number of viable cells was estimated using MTT assays as described (46). Statistical analysis was done using Student’s t test. Recombinant human MIS was collected from growth medium of Chinese hamster ovary cells transfected with the human MIS gene and purified as described (47). Immuno-affinity-purified MIS (35 nM) was added to the cells either alone or concurrently with R1881.

The dominant-negative (Smad1DN) construct in which the three serine residues at the carboxy-terminal SSVS motif were converted to alanines was a kind gift from Dr. Tongwen Wang (Benaroya Research Institute, VA Mason, Seattle, WA). LNCaP cells were stably transfected with either 12 µg vector or Smad1DN expression constructs along with 1 µg hygromycin-resistance plasmid. Hygromycin-resistant, Smad1DN-expressing clones were identified by Northern blot. Generation and characterization of the dominant-negative IB- (IBDN)-expressing LNCaP clones is described (9).

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 (8).

Luciferase Assays
The PSA-luciferase construct, a 6-kb PSA promoter-driven reporter, described in Refs. 48 and 49 , was a kind gift from Dr. Myles Brown (Dana Farber Harvard Cancer Center, Boston, MA). To test the effect of MIS on androgen-induced PSA promoter activity, LNCaP cells were maintained for 24 h in RPMI containing 5% charcoal-stripped serum and transfected with 0.45 µg PSA-luciferase, 1 µg CMV-MIS, or 1 µg Smad1DN or 1 µg IBDN expression constructs using Fugene-6. Twenty-four hours after transfection, cells were treated with either 0.1 or 10 nM R1881 as described in the figure, and luciferase activity was determined after 24 h. Untreated, vector-transfected LNCaP cells were used as controls. Total amount of transfected DNA was equalized by the addition of vector plasmid. Transfection efficiency was standardized by including 0.01 µg simian virus 40 (SV40)-Renilla luciferase reporter construct and luciferase assays were carried out using the dual luciferase assay kit (Promega, Madison, WI) as described in the user’s guide.

Antibodies and Western Blot Analysis
Proteins were extracted with radioimmunoprecipitation assay buffer and analyzed by Western blot as described (50). The E-cadherin antibody was purchased from Zymed (San Francisco, CA). The rabbit anti-phospho-Smad1 and Smad1 antibodies were purchased from Cell Signaling Technology (Beverly, MA); the rabbit anti-androgen receptor, -p50 and -p65 antibodies were from Santa Cruz (Santa Cruz, CA).

	ACKNOWLEDGMENTS

 
We thank Drs. Jose Teixeira and Alan Goldstein for critically reading this manuscript.

	FOOTNOTES

 
This work was supported by the Hershey Family Foundation and Survivors Walk for Prostate Cancer, Breast Cancer Research Grant from the Massachusetts Department of Public Health, the Harvard Medical School 50th Anniversary Scholars in Medicine Award, the Avon Breast Cancer Pilot Project Grant, and the Claflin Distinguished Scholar Award, and received partial support from the Dana-Farber Harvard Breast Cancer Specialized Program of Research Excellence from the National Cancer Institute (NCI) and National Institutes of Health (NIH)/NCI Grant CA89138 (to S.M.).

P.K.D. receives financial support from Ipsen/Biomeasure (Milton, MA), a company that is currently in the process of producing recombinant human MIS and Grants CA17373 and HD32112 from the NIH/NCI. None of the other authors have any disclosures to make.

First Published Online June 1, 2006

Abbreviations: ALK, Activin-like kinase; BMP, bone morphogenetic protein; DHT, dihydrotestosterone; DN, dominant negative; MIS, Mullerian inhibiting substance; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NFB, nuclear factor-B; PSA, prostate-specific antigen; SV40, simian virus 40.

Received for publication November 29, 2005. Accepted for publication May 24, 2006.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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