ß-Catenin Is Involved in Insulin-Like Growth Factor 1-Mediated Transactivation of the Androgen Receptor

Meletios Verras and Zijie Sun

Department of Urology and Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-5328

Address all correspondence and requests for reprints to: Dr. Zijie Sun, Department of Urology and Department of Genetics, R135, Edwards Building, Stanford University School of Medicine, Stanford, California 94305-5328. E-mail: zsun{at}stanford.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The androgen-signaling pathway is important for the growth and progression of prostate cancer cells. IGF-I and other polypeptide growth factors have been shown to be capable of induction of androgen receptor (AR) activation in the absence of, or at low levels of, ligand. It has been shown that IGF-I increases the cellular level of ß-catenin, an AR coactivator. In this study, we performed several experiments to test whether ß-catenin is involved in IGF-I-induced AR-mediated transcription. We demonstrate that IGF-I enhances the expression of endogenous prostate-specific antigen, an AR target gene, and elevates the level of cytoplasmic and nuclear ß-catenin in prostate cancer cells. Transfection of either wild-type or a constitutively active mutant of the IGF-I receptor augments AR-mediated transcription. An antisense construct of ß-catenin that decreases the cellular level of ß-catenin can reduce IGF-1 receptor-mediated enhancement of AR activity. Moreover, using a pulse-chase experiment, we showed that IGF-I enhances the stability of ß-catenin in prostate cancer cells. Our findings delineate a novel pathway for IGF-I in modulating androgen signaling through ß-catenin.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE FACT THAT androgen ablation is an effective treatment for the majority of metastatic prostate cancer patients indicates that androgen plays an essential role in regulating the growth of prostate cancer cells (1). Unfortunately, most patients develop androgen-insensitive prostate cancer within 2 yr, for which there is currently no effective treatment. Failure of androgen ablation suggests that there are alterations in androgen signaling in the tumor cells. Multiple mechanisms by which prostate cancer cells progress to androgen-insensitive stages have been proposed (2).

Previous experiments have shown that androgen receptor (AR) can be activated in cells treated with polypeptide growth factors in the absence of, or at low levels of androgens (3). IGF-I is the most efficient growth factor capable of ligand-independent activation of AR. However, the mechanisms by which IGF-I or other growth factors regulates AR-mediated transcription in prostate cells remain unclear. Recently, Playford et al. (4) reported that IGF-I enhances tyrosine phosphorylation of ß-catenin in human colorectal cancer cells, which results in dissociation of ß-catenin from E-cadherin complexes at the cell membrane. Induction by IGF-I also increases the stability of the ß-catenin protein (4). These results suggest a new link between ß-catenin and IGF-I-mediated cell growth and transformation.

Recently, we and others (5, 6, 7, 8) have demonstrated a specific protein-protein interaction between ß-catenin and AR. Through the interaction, ß-catenin augments AR-mediated transcription. ß-Catenin plays a pivotal role in cadherin-based cell adhesion and in the Wnt signaling pathway [see the review by Polakis (9)]. Most ß-catenin is located in the cell membrane where it is associated with the cytoplasmic region of E-cadherin, a transmembrane protein involved in homotypic cell-cell contacts (10). Smaller pools of ß-catenin are located in both the nucleus and cytoplasm, where the protein mediates Wnt signaling. The wnt signal and E-cadherin can modulate the cellular level of ß-catenin (6, 11). Accumulated ß-catenin translocates into the nucleus and forms transcriptionally active complexes with Tcf/LEF (12) or the AR (5, 6). AR can modulate Tcf/LEF-mediated cellular effects by binding to limiting amounts of ß-catenin, which may be critical during normal prostate development and tumor progression (13, 14).

Based on the facts that ß-catenin is an AR coactivator and that IGF-I affects AR-mediated transcription, we investigated the molecular mechanism by which IGF-I enhances AR activity. Particularly, we addressed whether the activation of AR by IGF-I in prostate cells is modulated by the cellular level of ß-catenin. In the human prostate cancer cell line, LNCaP, we show that IGF-I enhances AR-mediated transcription at a low level of androgen, and that it increases the level of cellular ß-catenin. Transfection of either wild-type or a constitutively active mutant of the IGF-I receptor (IGF-1R) augments AR-mediated transcription. Moreover, using an antisense construct of ß-catenin, we further demonstrate that a decrease in the cellular level of ß-catenin can reduce IGF-1R-mediated enhancement of AR activity. Furthermore, pulse-chase experiments demonstrate that IGF-I enhances the stability of the ß-catenin protein in prostate cancer cells. These findings delineate a novel mechanism by which IGF-I modulates androgen signaling in prostate cells and provides fresh insight into the role of IGF-I in the development of androgen-insensitive prostate cancer.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IGF-I Enhances the Expression of Prostate-Specific Antigen (PSA), a Target Gene of AR, in Prostate Cells
Previous experiments showed that AR can be activated in cells treated with growth factors in the absence of, or at low levels of ligand (3). IGF-I is the most efficient growth factor capable of ligand-independent activation of AR. To evaluate the effect of IGF-I in a biologically relevant setting, we tested whether IGF-I regulates expression of the PSA gene, an endogenous AR target, in the AR-positive prostate cancer cell line, LNCaP. Using real-time PCR, we first measured transcripts of PSA in LNCaP cells treated with different amounts of IGF-I. In the presence of 0.1 nM dihydrotestosterone (DHT), PSA expression was increased approximately 20 or 35% in LNCaP cells treated with 30 or 100 ng/ml of IGF-I, respectively, over that found in cells not treated with IGF-I (P < 0.05) (Fig. 1AGo). However, in the absence of DHT, the expression of PSA was not significantly affected by IGF-I. To confirm this finding, we examined the expression of PSA by conventional Northern blotting. As observed in the real-time PCR assays, an increase in PSA transcripts was found in the cells treated with 30 or 100 ng/ml of IGF-I (Fig. 1BGo). Using ß-actin as a reference gene, we showed an approximately 0.3- to 1-fold increase in PSA transcripts in the cells treated with 30 or 100 ng/ml of IGF-I, respectively (Fig. 1CGo). These results provide the first line of evidence that IGF-I is able to enhance endogenous AR-mediated transcription in prostate cancer cells in the presence of a low level of androgens.



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Fig. 1. IGF-I Enhances the Transcription of an Endogenous AR Target Gene, PSA

A, Expression levels of PSA mRNA were quantified using quantitative fluorescent real-time PCR. Total RNAs were isolated from LNCaP cells cultured in T-medium with or without 0.1 nM DHT, treated for 16 h with IGF-I or vehicle. RNA samples were first reverse-transcribed using random hexamers. Two specific primers selected from the regions around the translation initiation site or the stop codon of the PSA gene were used for amplification. PCR assays were performed with TaqMan PCR reagent Kits in the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). The levels of PSA mRNA were normalized by coamplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as described by the manufacturer (PE Applied Biosystems). B, Expression of the endogenous PSA transcripts was detected by a cDNA probe derived from the human PSA gene in LNCaP cells treated with IGF-I as described above. A ß-actin probe was used to confirm equal RNA loading. C, Densitometry of the Northern blot was performed, and the relative numbers are reported as PSA/ß-actin.

 
IGF-I Increases the Level of Cellular ß-Catenin in Prostate Cancer Cells
It has been shown that IGF-I can elevate the cellular level of ß-catenin in the human colon cancer cell line, C10 (4). ß-Catenin has been demonstrated to be an AR coactivator (5, 6). To determine whether ß-catenin is involved in IGF-I mediated AR transcription, we examined free cellular ß-catenin in prostate cancer cells as described previously (4). As shown in Fig. 2AGo, there was no significant change in the amount of ß-catenin in the cytoskeletal compartment (RIPA) of cells treated with IGF-I. However, there was a 2- to 4-fold increase in cytosolic ß-catenin in cells treated with 50 or 100 ng of IGF-I (Digi), respectively. In contrast, the level of cytosolic tubulin, used as a control, showed no significant difference in the treated and untreated cells.



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Fig. 2. IGF-I Signaling Enhances the Level of Free Cytosolic ß-Catenin

A, Both cytosolic (Digi) and cytoskeletal fractions (RIPA) were prepared from LNCaP cells as described in Materials and Methods. Both ß-catenin and tubulin proteins were analyzed by Western blotting with specific antibodies. B, Cytosolic fractions were isolated from LNCaP cells treated with different concentrations of growth factors. The levels of ß-catenin were analyzed by Western blotting. Tubulin was used as a control for protein loading. C, The endogenous ß-catenin proteins were analyzed in nuclear extracts isolated from LNCaP cells treated with or without IGF-I or DHT as labeled in the figure. The level of ß-catenin was detected by Western blotting with a specific ß-catenin antibody. H1, a nuclear protein, was used as a control for protein loading.

 
To further test whether the effect of IGF-I on ß-catenin is a specific event, we repeated the above experiments with LNCaP cells treated with different growth factors. As shown in the figure, a pronounced increase in cytosolic ß-catenin was observed in cells treated with IGF-I but not with EGF (Fig. 2BGo). With 100 ng/ml of IGF-II, a slight change of ß-catenin was also observed, suggesting a potential role of IGF-II in the regulation of cellular ß-catenin (see Discussion). In addition, we also performed transient transfection experiments using the PSA-promoter/reporter in LNCaP cells in the presence of the above growth factors. We observed that IGF-I is one of the most efficient growth factors in modulating AR-mediated transcription (data not shown), which is consistent with the previous report by Culig et al. (15).

Next, we examined whether IGF-I directly affects the translocation of ß-catenin to the nucleus. In the presence of 0.1 nM of DHT, IGF-I significantly increases the level of nuclear ß-catenin in LNCaP cells, whereas it only affects ß-catenin slightly in the absence of ligand (Fig. 2CGo). These data are consistent with our previous observation that IGF-I has a more pronounced effect in enhancing AR-mediated transcription in the presence of low level of androgens. The histone (H1) protein, used as a control, showed no change (Fig. 2CGo). Taken together, these results confirm the role of IGF-I in enhancing the translocation of ß-catenin into the nucleus, which agree with the previous studies showing the similar effect of IGF-I in enhancing nuclear ß-catenin in human colorectal cancer cells (4).

Overexpression of IGF-1R Enhances AR-Mediated Transcription
To confirm that IGF-I enhances AR activity, we tested the ability of either the wild-type IGF-1R or a constitutively activated receptor, IGF-1R-NM1, generated by deleting the entire extracellular domain of IGF-1R and fusing the remaining receptor (16), to enhance AR-mediated transcription. In cells cultured with 5% fetal calf serum, both the wild-type IGF-I and IGF-1R-NM1 augmented AR-mediated transcription from the PSA promoter in a dose-dependent manner (P < 0.05) (Fig. 3AGo). The IGF-1R-NM1 receptor showed a stronger augmentation of AR-mediated transcription than the wild-type receptor both in the absence and presence of androgens (P < 0.05). These results provide an additional line of evidence that IGF-I signaling plays a role in regulating AR-mediated transcription.



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Fig. 3. Overexpression of IGF-I Receptors Enhances AR-Mediated Transcription

A, Transient transfections were performed in LNCaP cells with 100 ng of PSA7kb-luc reporter and 10 or 30 ng of wild type (wt) or a constitutively active mutant (NM1) of IGF-I receptor and 25 ng of pcDNA3-ß-gal. The cells were incubated in T-medium with 5% charcoal-stripped FCS for 12 h, and then were treated with different concentrations of DHT for 18 h. Cell lysates were measured for luciferase and ß-gal activities. The data represent the mean ± SD of three independent samples. B, The transfection experiments were repeated in LNCaP cells under the same conditions as described above. Different amounts of the antisense ß-catenin plasmid were cotransfected into the cells. The relative light units (RLU) from individual transfections were normalized by measurement of ß-gal activity expressed from a cotransfected plasmid in the same samples. C, The levels of cytosolic ß-catenin were measured by Western blotting from LNCaP transfected with 30 ng of the IGF-I receptors and 30 ng of antisense ß-catenin plasmids (AS-ß-cat). D, One hundred nanograms of PSA7kb-luc reporter with or without 30 ng of the ß-catenin antisense plasmids were transfected into LNCaP cells. The cells were cultured in T-medium with or without 100 ng/ml of IGF-I in the presence or absence of DHT for 28–32 h. IGF-1R antibody (1 µg/ml) (catalog no. GR11, CalBiochem, San Diego, CA) or mouse normal IgG was added into cells 6 h after transfection. Luciferase and ß-gal activities were measured as described above.

 
To further demonstrate that the enhancement of AR activity by IGF-I receptors was mediated by ß-catenin, we repeated the above experiments with an antisense construct of ß-catenin (6). As shown in Fig. 3BGo, cotransfection of the antisense ß-catenin plasmid represses the enhancement of AR by both the wild-type and the mutant IGF-1R. With 30 ng of the antisense construct, AR activity was reduced 45% and 65% in cells transfected with the wild-type and mutant IGF-1R (P < 0.05), respectively. To evaluate the effectiveness of ß-catenin antisense constructs, we also measured the level of the cytosolic ß-catenin protein in the above samples. As expected, the antisense ß-catenin constructs reduced the levels of cytosolic ß-catenin (Fig. 3CGo), which correlated with the reduction in AR transcriptional activity in the cells. To further demonstrate the role of ß-catenin in IGF-I-induced AR activity, we examined whether the inhibition of ß-catenin expression can affect IGF-I-induced AR activity by using the ß-catenin antisense constructs. As shown in Fig. 3DGo, the IGF-I-induced AR transactivation of the PSA promoter is abrogated by cotransfection with the ß-catenin antisense constructs. In addition, we also demonstrate that the specific IGF-1R antibody, {alpha}IR3, effectively blocks the IGF-I mediated AR activity. Taken together, the above results demonstrate a direct involvement of ß-catenin in the IGF-I signaling modulated, AR-mediated transcription.

IGF-I Stabilizes ß-Catenin in Prostate Cancer Cells
It has been shown that IGF-I enhances the stability of the ß-catenin protein in human colorectal cancer cells (4). IGF-I inhibits glycogen synthase kinase-3ß (GSK3ß) by stimulating the phosphorylation of GSK3ß (17, 18). We therefore examined whether the IGF-I enhancement of the stability of ß-catenin in prostate cancer cells is mediated through GSK3ß. Because LiCl has been shown to repress GSK3ß (19), thereby stabilizing cellular ß-catenin, we measured the cytosolic level of ß-catenin in LNCaP cells treated with or without IGF-I. As observed previously, we saw an increase in ß-catenin in cells treated with IGF-I (Fig. 4AGo). In the presence of 50 mM LiCl, the samples isolated from cells treated with 100 ng/ml of IGF-I showed higher levels of ß-catenin than ones not treated with LiCl. We conclude that inhibition of GSK3ß can further enhance the levels of cellular ß-catenin induced by IGF-I in prostate cancer cells.



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Fig. 4. IGF-I Enhances the Stability of ß-Catenin

A, The cytosolic fraction was used to measure the free form of ß-catenin in LNCaP cells treated with different concentration of IGF-I in the presence or absence of 50 mM LiCl. B, LNCaP cells were pulsed with Tran35S-label and chased with medium containing an excess of cold methionine/cysteine for the indicated times. The cytosolic fractions isolated from cells were immunoprecipitated for ß-catenin. C, The results were analyzed by densitometry and expressed graphically as a percentage of the value at 0 h. The figure shows results of a single experiment, which was repeated once with similar results.

 
To demonstrate that the effect of IGF-I was on the stability of ß-catenin in prostate cancer cells, we performed pulse-chase experiments in LNCaP cells. Cells were pulsed with Tran35S label and chased in the presence or absence of IGF-I for 12 h. 35S-labeled ß-catenin proteins were immunoprecipitated from the cytosolic fractions and analyzed by SDS-PAGE. As shown in Fig. 4BGo, IGF-I enhances the stability of ß-catenin, increasing its half-life from 9–12 h. These results provide a direct line of evidence that IGF-I indeed affects the stability of ß-catenin in prostate cancer cells.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Induction of ligand-independent activation of AR by IGF-I and other polypeptide growth factors was initially observed several years ago (3). However, the mechanism(s) by which the growth factors induce AR-mediated transcription were still unknown. It has been shown that IGF-I increases the cellular level of ß-catenin in human colon cancer cells (4). In addition, ß-catenin has been demonstrated to interact with AR and to enhance AR-mediated transcription (5, 6, 8, 20). The results described here provide multiple lines of evidence demonstrating that ß-catenin is involved in the induction by IGF-I of AR-mediated transcription. This was demonstrated by experiments showing that IGF-I enhanced expression of PSA in prostate cells, that IGF-I increased the levels of ß-catenin at least in part by stabilizing the protein, and finally, that increasing the levels of IGF-1R in prostate cells enhances the level of AR-mediated transcription.

We showed that IGF-I enhances the expression of endogenous PSA transcripts in cells treated with low levels of the DHT by both real-time PCR and Northern blot experiments. These results are different from those of Culig et al. (3), who showed previously that induction of AR activity by IGF-I on reporters driven by the androgen response element (ARE)- or murine mammary tumor virus promoters was an androgen-independent effect. Obviously, there are several differences between our and their experiments. Although we do not know the exact reason(s) for these differences, we feel that our experiments, which examine the PSA transcripts, a natural, endogenous AR-target gene, are more sensitive and biologically relevant for assessing AR activity. It has been shown that the unbound AR forms a complex with heat-shock proteins (21), and upon binding to ligand, the AR dissociates from the heat-shock proteins and translocates into the nucleus (22). Our data agree with the previous observations, suggesting that the IGF-I-induced AR activity requires the nuclear translocation of AR upon binding to ligand, which is also supported by the recent observation that ß-catenin augmenting the AR-mediated activity is a nuclear effect and requires the nuclear translocation of AR in the presence of the ligand (6, 8).

LNCaP is currently the only well-characterized prostate cancer cell line that contains both functional AR and E-cadherin pathways and is responsive to IGF-I (23, 24, 25). However, it has been shown that there are a relatively low number of IGF-I receptors on these cells, approximately 1 x 104 receptors/cell (25). To examine the effect of having a more physiologically relevant number of receptors on the cells, we transfected wild-type IGF-1R and a constitutively activated mutant of IGF-1R into LNCaP cells. Both the wild-type and the mutant IGF-1R enhanced AR activity from the 7-kb PSA promoter in a dose-dependent manner, indicating that IGF signaling modulates AR-mediated transcription. We also performed transient transfection experiments with a luciferase reporter driven by a minimal promoter with two AREs in LNCaP cells. We only observed a moderate effect of IGF-1R on the ARE-reporter, in contrast to the PSA-reporter (data not shown). This result may suggest that other transcription factors that bind to sites adjacent to the ARE in the natural PSA-promoter may enhance the AR in response to IGF-I-mediated induction.

The cellular levels of ß-catenin are tightly regulated in normal cells. Mutations affecting the degradation of ß-catenin can result in the accumulation of the cellular ß-catenin to induce neoplastic transformation (26). Due to an abnormal cadherin-catenin interaction in the cell membrane, increasing the cytoplasmic and nuclear levels of ß-catenin as a consequence of loss of E-cadherin is also frequently observed in late stages of prostate cancer cells (27). In this current study, intriguingly, we demonstrated that IGF-I increases the cellular levels of ß-catenin in the prostate cancer cell line, LNCaP, which suggests a novel mechanism by which IGF-I modulates AR mediated transcription. Using both Western blot and pulse-chase assays, we further showed that an increase in ß-catenin levels by IGF-I in LNCaP cells is the result of stabilization of ß-catenin. Previous studies have shown that GSK3ß is one of the major components in the destruction complex that constitutively down-regulates the level of cellular ß-catenin. The signaling mediated by phosphoinositol 3-kinase (PI3K)/Akt can regulate GSK3ß through the phosphorylation of the protein (28). The tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), negatively regulates the PI3K pathway by blocking activation of Akt (29). Our observation that lithium chloride, an inhibitor of GSK3ß, enhances the stabilizing effect of IGF-I on ß-catenin suggesting a potential link between the IGF-I and PI3K/Akt signaling pathways in modulating Wnt/ß-catenin signaling. Our results are also consistent with an earlier report that showed that IGF-I can stabilize the ß-catenin protein in combination with lithium chloride in a human colon cancer cell line (4). Further studies of the interaction between IGF, PI3K/Akt, and Wnt/ß-catenin pathways may help us to understand their roles in cell-cell adhesion, cell migration, transformation, and tumor metastasis.

In this study, we showed that IGF-I enhances AR-mediated transcription and increases the levels of cellular ß-catenin. In addition, we also observed a slight increase in ß-catenin in cells treated with 100 ng/ml IGF-II (see Fig. 2BGo). This result suggests a possible role for IGF-II in the regulation of cellular ß-catenin, which is consistent with the recent report that showed that IGF-II induces rapid ß-catenin relocation to the nucleus during epithelium to mesenchyme transition (30). To further evaluate the roles of IGF-II and EGF in AR-mediated transcription, we also performed transient transfection experiments using the PSA-promoter/reporter in LNCaP cells. We observed a notable induction only by IGF-I in our experiments (data not shown), which is similar to the previous report by Culig et al. (3).

In conclusion, in this study we provide several lines of evidence linking IGF-I to the regulation of ß-catenin. Because ß-catenin has been identified as an AR coregulator, demonstration of a link between IGF-I and ß-catenin suggests a potential, novel mechanism by which IGF-I regulates prostate cancer cells in their progression to the androgen-insensitive stage. Further study of this linkage may help us to understand the roles of IGF-I signaling in prostate cancer pathogenesis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Real-Time PCR and Northern Blot Analysis
Total RNAs were isolated from LNCaP cells treated with IGF-I in the presence or absence of 0.1 nM DHT by using an RNAwiz kit (Ambion, Austin, TX), and RNA concentration was estimated from absorbance at 260 nm. Expression levels of PSA mRNA were quantified using quantitative fluorescent real-time PCR. RNA was first reverse-transcribed using random hexamers as described by the manufacturer (PE Applied Biosystems, Foster City, CA). Two specific primers selected from the regions around the translation initiation site or the stop codon of the PSA gene were used for amplification. PCR assays were performed with TaqMan PCR reagent Kits in the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). The levels of PSA mRNA were normalized by coamplification of GAPDH mRNA as described by the manufacturer (PE Applied Biosystems). Northern blotting assays were performed as described previously (31).

Cell Cultures and Transfections
The monkey kidney cell line, CV-1, was maintained in DMEM supplemented with 5% fetal calf serum (FCS) (HyClone, Denver, CO). An AR-positive prostate cancer cell line, LNCaP, was maintained in T-medium (Invitrogen Life Technologies, Carlsbad, CA) with 10% FCS. Transient transfections were carried out using LipofectAMINE 2000 (Invitrogen) as described previously (31). In the experiments with IGF-I and other growth factors (Sigma, St. Louis, MO), cells were usually cultured in T-medium for 16 h, and then were treated with different concentrations of growth factors for 20–24 h. For androgen induction experiments, cells were grown in T-medium with charcoal-stripped fetal calf serum (HyClone) for 16–24 h in the presence or absence of DHT.

Whole Cell and Nuclear Extracts
Both whole cell lysate and nuclear extracts were prepared as described previously (6, 32). The cytosolic or cytoskeletal fractions were prepared in digitonin lysis buffer [1% digitonin, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2] or in RIPA buffer [0.5% Nonidet P-40, 0.3% Triton X-100, 15 mM MgCl2, 5 mM EDTA, 150 mM NaCl, 50 mM Tris-HCl (pH 7.8)], respectively (4). Protein fractions for immunoblotting were boiled in sodium dodecyl sulfate-sample buffer and then resolved on a 10% SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane and probed with anti-ß-catenin antibody (catalog no. C19220, Transduction Labs, Lexington, KY). Proteins were detected using the ECL kit (Amersham, Arlington Heights, IL). The antibody against H1 (Santa Cruz Biotechnology, Santa Cruz, CA) or tubulin (Neomarker, Fremont, CA) was used for protein loading.

Plasmids
The reporter plasmid, pPSA7kb-luc, was provided by Dr. Jan Trapman (33). IGF-1R expression vectors were the kind gift of Dr. Weiqun Li (16). The antisense construct of human ß-catenin and the pcDNA3-FLAG-ß-catenin vector were generated as described previously (6).

Luciferase and ß-Galactosidase (ß-gal) Assay
Luciferase and ß-gal activities were measured as previously described (6, 32). The relative light units from individual transfections were normalized by measurement of ß-gal activity expressed from a cotransfected plasmid in the same samples. Individual transfection experiments were done in triplicate and the results are reported from representative experiments.

Pulse-Chase
LNCaP cells were transfected with wild-type pcDNA3-FLAG-ß-catenin. After 24 h of transfection, the cells were incubated with DMEM without L-methionine and L-cysteine (Invitrogen Life Technologies) for 1 h, and then pulse-labeled with 100 µCi of Tran 35S Label (ICN, Irvine, CA) for 30 min. The cells were washed twice with PBS and then chased by incubating in complete DMEM in the presence or absence of IGF-I (50 ng/ml) for various periods of time. The cells were lysed in RIPA and digitonin lysis buffers containing protease inhibitors. 35S-labeled ß-catenin protein was immunoprecipitated from the cytosolic fractions using an anti-ß-catenin rabbit polyclonal antibody (H-102; Santa Cruz Biotechnology) and analyzed by SDS-PAGE.

Statistical Analysis
The difference in the values between two groups was analyzed using the Student’s t test. P < 0.05 was considered statistically significant.


    ACKNOWLEDGMENTS
 
We are especially grateful for the DNA plasmids supplied by Drs. Weiqun Li (National Cancer Institute, Bethesda, MD) and Jan Trapman (Erasmus University, Rotterdam, The Netherlands). We thank Dr. Geoffrey Kitchingman for a critical reading of our manuscript.


    FOOTNOTES
 
This work was supported by National Institutes of Health Grants CA70297, CA87767, and DK61002, and the Department of Army Prostate Cancer grants (DAMD17-00-1-0296 and DAMD17-03-1-0090), and California Cancer Research Grant 03-00163VRS-30083.

First Published Online October 28, 2004

Abbreviations: AR, Androgen receptor; ARE, androgen response element; DHT, dihydrotestosterone; FCS, fetal calf serum; ß-gal, ß-galactosidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK3ß, glycogen synthase kinase-3ß; H1, histone; IGF-1R, IGF-I receptor; PI3K, phosphoinositol 3-kinase; PSA, prostate-specific antigen.

Received for publication May 20, 2004. Accepted for publication October 19, 2004.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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