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Requirement of Androgen-Dependent Activation of Protein Kinase C for Androgen-Dependent Cell Proliferation in LNCaP Cells and Its Roles in Transition to Androgen-Independent Cells

Department of Urology (T.I., T.Yo., Y.Sh., T.Ko., T.Ya., T.Se., T.Ka., E.N., O.O.), Kyoto University, Graduate School of Medicine, and Anatomical Center of Kyoto University (Y.T.), Graduate School of Medicine, 54 Kawaharacho, Shogoin Sakyo-ku Kyoto 606-8507, Japan

Address all correspondence and requests for reprints to: Eijiro Nakamura, M.D., Ph.D., Department of Urology, Kyoto University Graduate School of Medicine, 54 Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: hap{at}kuhp.kyoto-u.ac.jp.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
A cell line that we designed, AILNCaP, proliferated in androgen-depleted medium after emerging from long-term androgen-depleted cultures of an androgen-sensitive prostate cancer cell line, LNCaP. Using this cell line as a model of progression to androgen independence, we demonstrated that the activity of the mammalian target of rapamycin/p70 S6 kinase transduction pathway is down-regulated after androgen depletion in LNCaP, whereas its activation is related to transition of this cell line to androgen-independent proliferation. Kinase activity of protein kinase C is regulated by androgen stimulation in LNCaP cells, whereas it is activated constitutively in AILNCaP cells under androgen-depleted conditions. Treatment with a protein kinase C pseudosubstrate inhibitor reduced p70 S6 kinase activity and cell proliferation in both cell lines. We identified that both protein kinase C and p70 S6 kinase were associated in LNCaP cells and this association was enhanced by the androgen stimulation. We examined the expression of phospho-protein kinase C and phospho-p70 S6 kinase in hormone-naive prostate cancer specimens and found that the expression of both kinases was correlated with each other in those specimens. Significant correlation was observed between the expression of both kinases and Ki67 expression. Most of the prostate cancer cells that survived after prior hormonal treatment also expressed both kinases. This is the first report that shows the significance of this pathway for both androgen-dependent and -independent cell proliferation in prostate cancer. Our data suggest that protein kinase C/mammalian target of rapamycin/S6 kinase pathway plays an important role for the transition of androgen-dependent to androgen-independent prostate cancer cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
SINCE DEMONSTRATION BY Huggins and Hodges in the early 1940s that growth and survival of the prostate gland depends upon androgens (1), the mainstay of treatment for advanced prostate cancer has been androgen ablation. However, the vast majority of patients ultimately relapse after a period of initial response to this therapy, developing androgen-independent prostate cancer. Molecular mechanisms involved in prostate-cell proliferation in response to androgen, as well as emergence of androgen-independent prostate cancer, are under active investigation (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14).

The LNCaP cell line was established from supraclavicular lymph node metastasis of human prostate cancer (15). Regardless of its expression of a promiscuous gain-of-function mutant androgen receptor (AR) in LNCaP cells, androgens regulate their growth and expression of prostate-specific genes, such as the prostate-specific antigen (16). Due to this androgen-sensitive characteristic, the LNCaP cell line has become a widely used model for in vitro prostate cancer research. Although acute androgen ablation results in growth arrest without inducing apoptosis in the cell lines, long-term androgen ablation transforms a subpopulation to androgen-independent clones (17). Thus, a number of androgen-independent LNCaP sublines have been developed in different laboratories (3, 17, 18, 19, 20).

During the past decade, many reports have demonstrated that TOR (target of rapamycin) participates importantly in controlling cell growth, proliferation, and metabolism (21, 22). Regulation by TOR extends to a wide array of cellular functions including translation, transcription, mRNA turnover, protein stability, actin cytoskeletal organization, and autophagy (21, 22, 23).

The best-characterized downstream targets of mammalian target of rapamycin (mTOR) in mammalian cells include two signaling pathways that regulate translation: the 70-kDa ribosomal protein S6 kinase (S6K) pathway, and the eukaryotic translation initiation factor 4E-binding protein (4EBP1) pathway (22). Both 4E-BP and S6K pathways contribute to regulation of cell-cycle progression by TOR (22, 24). S6K is the well-known ribosomal protein S6 kinase in mammalian cells (22, 24). Microinjection of neutralizing anti-S6K antibodies can block mitogen-induced progression from the G1 to the S phase of the cell cycle (25). Phosphorylation of S6K contributes to an increase in kinase activity, which increases degrees of phosphorylation in ribosomal S6 polypeptides (26). S6 phosphorylation stimulates translation of mRNAs with 5'-terminal oligopyrimidine tracts (26). These terminal oligopyrimidine-containing mRNAs often code for components of the protein synthesis apparatus (26). Hence, S6K enhances the translational capacity of cells.

Multiple additional effectors for S6K include Cdc42 (27), Rac (27), 3-phosphoinositide-dependent protein kinase-1 (PDK-1) (28), and atypical protein kinase C (aPKC) (29, 30). In coexpression experiments, a kinase-inactive mutant of the aPKC isoform PKC antagonized activation of S6K by epidermal growth factor, PDK-1, and activated Cdc42 and phosphatidylinositol 3-kinase (PI3K) (29). This suggested that PKC participates in activation of multimetric PI3K-S6K signaling complexes. PKC has been identified as a downstream target of PI3K (31, 32). Differing from conventional and novel classes of PKCs, PKC does not require diacylglycerol or calcium for activation (33, 34, 35). Additionally, PKC has been suggested to be involved in several growth-related processes (36), and PDK-1 has been suggested as an activator of PKC (31, 32).

In this study, we established a prostate cancer cell line, AILNCaP, that proliferated under androgen-depleted conditions after emerging from long-term, androgen-depleted culture of an androgen-sensitive prostate cancer cell line, LNCaP. We used this model, which mimics the situation in patients subjected to androgen ablation therapy who eventually became refractory to treatment, to investigate the role of intracellular signaling pathways in progression to androgen independence in prostate cancer.

We demonstrated that the mTOR/S6K signaling pathway is activated in an androgen-dependent manner in LNCaP cells but is activated constitutively in AILNCaP cells under androgen-depleted conditions. We also found that phosphorylation of PKC is regulated by androgen stimulation in LNCaP cells while being active in AILNCaP cells even under androgen-depleted conditions. Using a myristoylated PKC pseudosubstrate inhibitory peptide, which is specific for atypical PKC, we demonstrated that PKC is required for S6K activation and cell proliferation in both LNCaP and AILNCaP cells.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Characterization of Androgen-Independent Cell Lines AILNCaP
As previously described (37, 38), short-term androgen deprivation in LNCaP cells results in cell-cycle arrest in G₁ rather than apoptosis. Here, instead of cell-cycle arrest, AILNCaP cells could proliferate even though they were cultured in androgen-deprivation medium, representing androgen-independent cell proliferation (Fig. 1A). To identify the mechanisms that may be responsible for androgen-independent proliferation of these AILNCaP cells, we first examined whether mutations of the AR gene in addition to T877A spontaneously occurred in AILNCaP. We did not find any AR mutations except for T877A, which also was present in the parental LNCaP cells (data not shown). Next we used Western blotting to compare AR expression between these cell lines, and it was revealed that AR expression in AILNCaP cells increased 2.2-fold over that in LNCaP cells (Fig. 1B). In fact, AILNCaP cells expressed prostate-specific antigen (PSA) in response to R1881 stimulation, but needs much higher dose of R1881 stimulation for PSA expression both in mRNA and protein levels than LNCaP cells did (Fig. 1C). We then examined whether low-dose androgen increases the cell proliferation of AILNCaP cells. Our results showed that low-dose androgen stimulation such as 0.001 nM of R1881 did not induce up-regulation of S phase in AILNCaP cells and the proportion of cells in S phase was most abundant under androgen depleted conditions (Fig. 1D). On the other hand, LNCaP cells entered S phase with 0.1 nM of synthetic androgen (R1881) stimulation after 96-h culture in charcoal-stripped fetal bovine serum (CSFBS) (Fig. 1D). To further examine the role of an extremely low concentration of androgen retained in CSFBS in the proliferation of AILNCaP cells, those cells were cultured in 1 µM of bicalutimide, which could inhibit cell proliferation of LNCaP cells under normal serum. Our results demonstrate that AILNCaP cells showed no attenuation in cell proliferation at 1 µM of bicalutamide treatment (Fig. 1E). Hence, AILNCAP cells do not scavenge residual androgen from the CSFBS medium. These results indicate that hypersensitivity to androgen may not cause the androgen-independent cell proliferation of AILNCaP cells.

View larger version (40K): [in this window] [in a new window]   Fig. 1. Effect of Androgen Deprivation and Stimulation in LNCaP Cells and AILNCaP Cells A, LNCaP cells were cultured in phenol red-free RPMI +10% CSFBS for 96 h and then 1 x 105 cells were seeded into 12-well plates (d 0). After 24 h, they were grown in 10% normal serum (FBS) (filled squares) or 10% CSFBS (open squares) for the indicated days. Cells were counted in a hemocytometer in quadruplicate. AILNCaP cells growing in phenol red-free RPMI + 10% CSFBS (open diamonds) also were analyzed. B, After 24 h in RPMI + 10% FBS, LNCaP cells were deprived of androgen in phenol red-free RPMI + 10% CSFBS for 96 h and total cell lysates were analyzed. AILNCaP cells in growth phase cultured in phenol red-free RPMI + 10% CSFBS also were analyzed, as were LNCaP cells in growth phase in RPMI + 10% FBS. Total cell extracts were analyzed for AR by Western blotting. The number under each lane of ß-actin expression indicates the relative intensity of each AR expression normalized to ß-actin expression. C, Dose-dependent expression analysis of PSA. After 24 h in RPMI + 10% FBS, LNCaP cells were depleted of androgen by culture in phenol red-free RPMI + 10% CSFBS for 96 h; then the indicated dose of synthetic androgen (R1881) was provided for 24 h. After 48 h in phenol red-free RPMI + 10% CSFBS, AILNCaP cells were treated with the indicated dose of synthetic androgen (R1881) for 24 h. Total cell extracts or RNA were analyzed for PSA by quantitative (upper panel) and conventional RTPCR (middle panel) or by Western blotting (lower panel). D, Percentages of LNCaP cells and AILNCaP cells in S phase determined by flow cytometry. LNCaP cells were cultured in phenol red-free RPMI + 10% CSFBS for 96 h and then the indicated dose of synthetic androgen (R1881) with 10% CSFBS or 10% FBS without R1881 was provided for 84 h. AILNCaP cells were cultured in phenol red-free RPMI +10% CSFBS and stimulated with indicated concentration of R1881 for 84 h before trypsinization and fixation. Values represent the mean ± SD derived from three independent experiments. All experiments were repeated at least three times. E, LNCaP cells were cultured in RPMI +10% FBS and then 1 x 105 cells were seeded into 12-well plates. AILNCaP cells were cultured in phenol red-free RPMI + 10% CSFBS and 1 x 105 cells seeded into 12-well plates also were analyzed. After 24 h, they were grown in RPMI +10% FBS (LNCaP) or phenol red-free RPMI +10% CSFBS (AILNCAP) with or without 1 µM of bicalutamide for 120 h. Cells were counted in a hemocytometer in quadruplicate. Data in panels represent the mean cell numbers relative to those without bicalutamide treatment ± SD (error bars).

View larger version (53K): [in this window] [in a new window]   Fig. 2. The mTOR/S6K Signaling Pathway Is Regulated by Androgen Stimulation in LNCaP Cells and Is Activated in AILNCaP Cells without Androgen Stimulation A and C, After 24 h culture in RPMI + 10% FBS, LNCaP cells were deprived of androgen in phenol red-free RPMI + 10% CSFBS for the time indicated, and total cell lysates were analyzed. AILNCaP cells in growth phase cultured in phenol red-free RPMI + 10% CSFBS also were analyzed, as were LNCaP cells in growth phase in RPMI + 10% FBS. Total cell extracts were analyzed for Erk phospho-T202/Y204 (p-Erk), p38MAPK phospho-T180/Y182 (p-p38MAPK), S6K phospho-T389 (p-S6KT389), S6K phospho-S371 (p-S6KS371), S6 phospho-S235/236 (p-S6), Erk, p38MAPK, S6K, and S6. The number under each lane of ß-actin expression indicates the relative intensity of each phospho-Erk (A) or phospho-S6KT389 (C) expression normalized to ß-actin expression. B, Upper panel, AILNCaP cells cultured in phenol red-free RPMI + 10% CSFBS were seeded 1 x 105 cells per well into 12-well plates (d 0). After 24 h, they were grown in phenol red-free RPMI + 10% CSFBS in the absence (filled squares) or presence of 10 µM PD98059 (open diamonds) for the indicated days. Cells were counted in a hemocytometer in quadruplicate. Left lower panel, Percentages of AILNCaP cells in S phase (white bars) and sub-G1 fraction (black bars) determined by flow cytometry. AILNCaP cells were cultured in phenol red-free RPMI + 10% CSFBS in the absence (–) or presence (+) of 10 µM PD98059 for72 h before trypsinization and fixation. Values represent the mean ± SD derived from three independent experiments. Right lower panel, After 48 h in phenol red-free RPMI + 10% CSFBS, AILNCaP cells were treated with 10 µM of PD98059 for 2 h. Total cell extracts were analyzed for Erk phospho-T202/Y204 (p-Erk) and Erk. D, After 24 h in RPMI + 10% FBS, LNCaP cells were deprived of androgen in phenol red-free RPMI + 10% CSFBS for 96 h; then the indicated final concentration of synthetic androgen (R1881) was added for 24 h. Total cell extracts were analyzed for S6K phospho-T389 (p-S6KT389) and for S6K. The number under each lane of ß-actin expression indicates the relative intensity of each phospho-S6KT389 expression normalized to ß-actin expression. E, S6K activity assays were carried out after growth of LNCaP cells in RPMI + 10% FBS as well as AILNCaP cells in phenol red-free RPMI + 10% CSFBS. LNCaP cells deprived of androgen in phenol red-free RPMI +10% CSFBS for 96 h and then stimulated with 10 nM of synthetic androgen (CSFBS+, R1881+), or sham-stimulated (CSFBS+, R1881–), for 24 h were also analyzed. S6K activity results are expressed as mean activities relative to those in androgen-depleted LNCaP cells after 120 h (CSFBS+, R1881–), ± SD (error bars) of (n) determinations. Asterisks indicate P < 0.01 for comparisons of S6K fold activities of LNCaP cells under CSFBS (CSFBS+, R1881–) values vs. others indicated. All experiments were repeated at least three times.

mTOR/S6K Signaling Pathway Is Required for Cell Proliferation in AILNCaP Cells
One main regulator of T389 phosphorylation that is crucial to activation of S6K (26, 42) is mTOR (43). We therefore treated cells with a specific inhibitor for mTOR, rapamycin, in the presence or absence of androgen to test whether androgen stimulation independently regulates S6K. This treatment abolished T389 phosphorylation of S6K in LNCaP cells regardless of androgen stimulation (Fig. 3A), suggesting that androgen acted upstream of mTOR. Moreover, treatment with rapamycin also reduced T389 and S371 phosphorylation of S6K in AILNCaP cells (Fig. 3B). Additionally, treatment with rapamycin partially inhibited the G1/S transition in LNCaP cells under normal serum and AILNCaP cells subjected to androgen deprivation. All these results suggest that activation of mTOR/S6K signaling pathway was also required for androgen-dependent cell proliferation in LNCaP cells and androgen-independent cell proliferation in AILNCaP cells (Fig. 3C).

View larger version (41K): [in this window] [in a new window]   Fig. 3. Action of mTOR/S6K Is Essential for Cell Proliferation in Prostate Cancer Cells A, After 24 h in RPMI + 10% FBS, LNCaP cells were cultured in phenol red-free RPMI + 10% CSFBS with or without 0.1 nM synthetic androgen R1881 for 48 h. LNCaP cells then were treated with 0.1 nM or 1 nM rapamycin for 24 h in the presence or absence of 0.1 nM R1881. Total cell extracts were analyzed for S6K phospho-T389 (p-S6KT389) and for S6K. B, After 48 h in phenol red-free RPMI + 10% CSFBS, AILNCaP cells were treated with 0.1, 0.5, 1, 5, or 10 nM rapamycin for 24 h. Total cell extracts were analyzed for S6K phospho-T389 (p-S6KT389) and S6K phospho-S371 (p-S6KS371). C, Cell-cycle distribution was determined by flow cytometry. After 24 h in RPMI + 10% FBS (LNCaP) or in phenol red-free RPMI + 10% CSFBS (AILNCaP), LNCaP cells and AILNCaP cells were cultured with or without rapamycin (1 nM) for 72 h. Left, Representative histograms of each condition. Right, Data in a panel represent the mean ± SD (error bars) of three independent experiments. These results are representative of three independent experiments.

View larger version (55K): [in this window] [in a new window]   Fig. 4. Activity and Expression of PDK-1/Akt and TSC2 Cannot Explain Progression to Androgen Independence in LNCaP Cells A, After 24 h in RPMI + 10% FBS, LNCaP cells were deprived of androgen in phenol red-free RPMI + 10% CSFBS for 96 h and total cell lysates were analyzed. AILNCaP cells in growth phase cultured in phenol red-free RPMI + 10% CSFBS also were analyzed, as were LNCaP cells in growth phase in RPMI + 10% FBS. Total cell extracts were analyzed for PDK-1 phospho-S241 (p-PDK-1), Akt phospho-S473 (p-Akt), PDK-1, and Akt. The number under each lane of ß-actin expression indicates the relative intensity of each phospho-PDK-1 and phospho-Akt expression normalized to ß-actin expression. B, Expression and phosphorylation of TSC2 were examined after culture under conditions described for panel A. Total cell extracts were analyzed for TSC2 phospho-T1426 (p-TSC2) and TSC2. C, After 48 h in phenol red-free RPMI + 10% CSFBS, both LNCaP cells and AILNCaP cells were treated for 30 min with or without 10 and 50 µM LY294002 or 10 and 50 nM wortmannin followed by culturing in fresh phenol red-free RPMI + 10% CSFBS for 120 min before harvesting. Left, Total cell extracts were analyzed for Akt phospho-S473 (p-Akt), S6K phospho-T389 (p-S6KT389), S6 phospho-S235/236 (p-S6), Akt, S6K, and S6. Right, A densitometric analysis of phospho-Akt and phospho-S6KT389 levels normalized to ß-actin expression in each condition (Ctrl: no stimulation, LY10: 10 µM LY294002, LY50: 50 µM LY294002, W10: 10 nM wortmannin, W50: 50 nM wortmannin). These results are representative of three independent experiments.

Based on these results, we investigated whether phosphorylation of S6K is PI3K sensitive, especially under androgen-depleted conditions in prostate cancer cells. Indeed, under androgen-depleted conditions, treatment with PI3K inhibitors, LY294002 or wortmannin, reduced phosphorylation of S6K (Fig. 4C). Interestingly, there was a difference in the level of S6K phosphorylation on threonine 389 with treatment of PI3K inhibitors, which was also reflected by the extent of S6K target protein S6 phosphorylation between LNCaP and AILNCaP cells (Fig. 4C). On the other hand, some discrepancies were evident between degree of attenuation of Akt phosphorylation and S6K phosphorylation by PI3K inhibitors in both cell lines (Fig. 4C). Considering that the phosphorylation status of Akt showed no significant difference between LNCaP cells and AILNCaP cells under androgen deprivation (Fig. 4C), other kinases downstream of PI3K as opposed to Akt might play major roles in mTOR/S6K activation in these cell lines.

Androgen Stimulation in LNCaP Cells Activates PKC, which Is Active in AILNCaP Cells without Androgen Stimulation
Recent studies suggested that PKC is an upstream activator of S6K (29, 30) and that PKC/S6K signaling pathway regulates cell proliferation of prostate cancer in a murine prostate cancer model (48). So, we examined phosphorylation of the threonine residue (Thr410) of PKC in LNCaP cells and in AILNCaP cells. In LNCaP cells, androgen deprivation induced PKC dephosphorylation of Thr410 (Fig. 5A, upper panel). In contrast, phosphorylation at Thr410 could be seen in AILNCaP cells even under androgen-deprived states (Fig. 5A). Furthermore, androgen stimulation up-regulated the degree of phosphorylation of PKC in LNCaP cells (Fig. 5A). In fact, AILNCaP cells retained higher PKC kinase activity than that in LNCaP cells under androgen-depleted conditions (Fig. 5B). Additionally, PKC kinase activity was partially restored by stimulation with synthetic androgen in LNCaP cells (Fig. 5B).

View larger version (53K): [in this window] [in a new window]   Fig. 5. Activity of PKC Is Regulated by Androgen Stimulation in LNCaP Cells and Is Activated in AILNCaP Cells without Androgen Stimulation A, Upper panel, Expression and phosphorylation of PKC under the conditions described for Fig. 4A were determined by immunoblotting. Total cell extracts were analyzed for PKC phospho-T410 (p-PKC) and for PKC. The number under each lane of ß-actin expression indicates the relative intensity of each phospho-PKC expression normalized to ß-actin expression. Lower panel, After 24 h in RPMI + 10% FBS, LNCaP cells were deprived of androgen in phenol red-free RPMI + 10% CSFBS for 96 h; then the indicated final concentration of synthetic androgen (R1881) was added for 24 h. Total cell extracts were analyzed for PKC phospho-T410 (p-PKC), S6K phospho-S389 (p-S6KT389), PKC, and S6K. The number under each lane of ß-actin expression indicates the relative intensity of each phospho-PKC expression normalized to ß-actin expression. B, PKC activity assays were carried out after growth of LNCaP cells in RPMI + 10% FBS as well as AILNCaP cells in phenol red-free RPMI + 10% CSFBS. LNCaP cells deprived of androgen in phenol red-free RPMI +10% CSFBS for 96 h and then stimulated with 10 nM of synthetic androgen (CSFBS+, R1881+), or sham-stimulated (CSFBS+, R1881–), for 24 h were also analyzed. PKC activity results are expressed as mean activities relative to those in androgen-depleted LNCaP cells after 120 h (CSFBS+, R1881–), ± SD (error bars) (n = 5). Asterisks indicate P < 0.01 for comparisons of PKC fold activities of LNCaP cell under CSFBS (CSFBS+, R1881–) values vs. others indicated. C, Upper panel, HEK 293 cells were cotransfected with HA-S6K (1 µg) and PKC-myc (5 µg) per 6-cm dish. The cells were starved in serum-free DMEM for 24 h and then stimulated with EGF (100 ng/ml) for 30 min before harvesting. LNCaP cells were cotransfected with HA-S6K (6 µg) and PKC-myc (18 µg) per 10-cm dish. The cells were culture in DMEM supplemented with 10% FBS for 48 h before harvesting. PC3 cells were cotransfected with HA-S6K (2 µg) and PKC-myc (6 µg) per 6-cm dish. The cells were starved in serum-free DMEM for 24 h and then stimulated with EGF (100 ng/ml) for 30 min before harvesting. Lower panel, LNCaP cells were cotransfected with HA-S6K (6 µg) and PKC-myc (18 µg) per 10-cm dish. LNCaP cells were cultured with phenol red-free RPMI +10% CSFBS with 10 nM of synthetic androgen (R1881+), or without androgen (R1881–), for 48 h before harvesting. PKC-myc was immunoprecipitated (IP) with an anti-myc antibody. For control experiments, the cell lysates were incubated with protein G-Sepharose conjugated with control mouse IgG (Ctrl Ab). Coimmunoprecipitating HA-S6K was detected using a S6K-specific polyclonal antibody (Santa Cruz). Total cell lysates were analyzed for HA-S6K using HA antibody (Covance). All experiments were independently repeated two to three times. D, After 24 h in RPMI + 10% FBS, LNCaP cells were deprived of androgen in phenol red-free RPMI + 10% CSFBS for 96 h. With or without pretreatment with 10 µM bicalutamide for 24 h, LNCaP cells were treated with or without 10 nM synthetic androgen (R1881) for another 24 h in phenol red-free RPMI + 10% CSFBS. Total cell extracts were analyzed for PKC phospho-T410 (p-PKC), S6K phospho-S389 (p-S6KT389), PKC, S6K, and PSA. The number under each lane of ß-actin expression indicates the relative intensity of each phospho-PKC expression and phospho-S6KT389 normalized to ß-actin expression. These results are representative of three independent experiments.

PKC Activity Is Required for the Cell Proliferation of LNCaP and AILNCaP Cells
To elucidate whether PKC activity is required for phosphorylation of S6K, treatment with a myristoylated PKC pseudosubstrate peptide, which is a proven specific inhibitor, was performed in LNCaP cells during androgen stimulation and in AILNCaP cells under androgen deprivation. Inhibition of PKC activity induced a decrease in the expression of both phosphorylated (Thr-389) and total S6K. The same trend was observed in downstream target S6, and the abundance of cyclin D1 signal was reduced in both cell lines (Fig. 6A). Additionally, cell cycle analysis revealed that 20 µM myristoylated PKC pseudosubstrate peptide not only inhibited the G1/S transition but also induced sub-G1 fraction in both cells, which strongly suggested the existence of apoptotic cells (Fig. 6B). This finding was confirmed by morphological analyses showing that around 10% of the cells presented the nuclei that were condensed or fragmented (Fig. 6C).

View larger version (39K): [in this window] [in a new window]   Fig. 6. Activity of the mTOR/S6K Signaling Pathway in LNCaP Cells Is Regulated by Activity of PKC, which Is Required for Androgen-Independent Cell Proliferation A, After 24 h in phenol red-free RPMI + 10% CSFBS, LNCaP cells were cultured for another 24 h with R1881 (10 nM) stimulation. Thereafter, LNCaP cells were treated with 10, 20, or 50 µM myristoylated PKC pseudosubstrate inhibitor (PKC PS) for 120 to 150 min. AILNCaP cells in phenol red-free RPMI + 10% CSFBS without R1881 also were treated with 10, 20, and 50 µM myristoylated PKC pseudosubstrate inhibitor (PKC PS) for 120–150 min. Total cell extracts were analyzed for S6K phospho-T389 (p-S6KT389), S6 phospho-S235/236 (p-S6), S6K, S6, and cyclin D1 (CCND1). The number under each lane of ß-actin expression indicates the relative intensity of each phospho-S6KT389 expression normalized to ß-actin expression. B, Cell-cycle distribution was determined by flow cytometry. After 24 h in RPMI + 10% FBS, LNCaP were treated with (+) or without (–) 20 µM myristoylated PKC pseudosubstrate inhibitor (PKC PS) for24 h. After 48 h in phenol red-free RPMI + 10% CSFBS, AILNCaP were treated with (+) or without (–) 20 µM myristoylated PKC pseudosubstrate inhibitor (PKCPS) for24 h. Data in panels represent the mean ± SD (error bars) of three independent experiments. C, Apoptosis was detected morphologically by using Hoechst 33342. LNCaP cells and AILNCaP cells treated with 20 µM myristoylated PKC pseudosubstrate inhibitor for 24 h were collected. The cells were treated with 10% formalin neutral buffer solution, followed by rinsing with phosphate-buffer saline, and Hoechst 33342 was added at a final concentration of 0.167 µg/µl and incubated for 20 min at room temperature in the dark. Cells aliquots were placed on slides and a fluorescent microscope was used to count 200 fluorescent cells per condition. Nuclear fragmentation and chromatin condensation were scored as dead. Representative morphology after treatment with myristoylated PKC pseudosubstrate inhibitor was represented. Left upper panels, There was no increase in chromatin condensation in LNCaP cells and AILNCaP cells treated with vehicles [PKCPS (–)]. Left lower panels, White arrows indicate cells with chromatin condensation and white arrowheads indicate nuclear fragmentation [PKCPS (+)]. Right panel, Graph represents three independent experiments in which 200 fluorescent cells were counted and scored for chromatin condensation and nuclear fragmentation.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Activity of the mTOR/S6K Signaling Pathway Is Regulated by PKC in AR-Negative Prostate Cancer Cell Lines and this Signaling Pathway Is Required for their Cell Proliferation A, After 24 h plating in RPMI + 10% FBS, PC3, and DU145 cells were treated with 10, 20, or 50 µM myristoylated PKC pseudosubstrate inhibitor (PKCPS) for 150 min. Total cell extracts were analyzed for S6K phospho-T389 (p-S6KT389), S6 phospho-S235/236 (p-S6), S6K, S6, and cyclin D1 (CCND1). The number under each lane of ß-actin expression indicates the relative intensity of each phospho-PKC expression and phospho-S6KT389 normalized to ß-actin expression. B, Apoptosis was detected morphologically by using Hoechst 33342. PC3 cells and DU145 cells treated with 50 µM myristoylated PKC pseudosubstrate inhibitor for 12 h were collected. The cells were treated with 10% formalin neutral buffer solution, followed by rinsing with phosphate-buffer saline, and Hoechst 33342 was added at a final concentration of 0.167 µg/µl and incubated for 20 min at room temperature in the dark. Cell aliquots were placed on slides and a fluorescent microscope was used to count 200 fluorescent cells per condition. Nuclear fragmentation and chromatin condensation were scored as dead. Graph represents three independent experiments in which 200 fluorescent cells were counted and scored for chromatin condensation and nuclear fragmentation.

Phospho-PKC Expression Is Related to Phospho-S6K Expression in Human Prostate Cancer Specimens

In the hormone-naive prostate cancer specimens, 75% of spots (138 of 184 spots) were positively stained with both phospho-PKC and phospho-S6K antibodies (Table 1). Case 1 and case 2 show the representative specimens with positively or negatively staining of both antibodies (Fig. 8). Spearman’s rank order correlation analysis revealed the statistical significance of the modest correlations between the expression of phospho-PKC and that of phospho-S6K in these specimens (P < 0.01, = 0.663) (Table 1). Furthermore, significant elevation of Ki67 labeling indexes was observed in specimens that were positively stained by both antibodies (total of 138 spots) in comparison with the rest of specimens (total of 46 spots) (P < 0.001). These results indicate that activation of PKC/S6 kinase pathway is associated with cell proliferation of hormone-naive prostate cancer cells in vivo. There was no correlation with Gleason grade and the expression of both kinases.

View this table: [in this window] [in a new window]   Table 1. Relationship between Phospho-PKC and Phospho-S6K Stainings of Each Cancer Spot in Tissue Microarray Derived from Clinically Localized Horomone-Naive (n = 67) (A) or Hormone-Treated (n = 12) (B) Prostate Cancer Patients1

View larger version (74K): [in this window] [in a new window]   Fig. 8. Expression of Phospho-PKC and Phospho-S6K Strongly Associates in Human Prostate Cancer Specimens Upper panels, Representative immunohistochemical staining of human prostate cancer specimens from three individual patients with hormone-naive (Cases 1 and 2) or hormone-treated prostate cancer (Case 3) for AR (A, E, and I), Ki67 (B, F, and J), phospho-PKC (C, G, and K), and phospho-S6K (D, H, and L) by each specific antibody. Sections were counterstained with hematoxylin. (Case 1; A–D) Spots with Gleason 3 cancer showing moderate-to-strong (++) staining of phospho-PKC and phospho-S6K. (Case 2; E–H) Spots with Gleason 3 cancer showing negative (–) staining of phospho-PKC and weak (+) of phospho-S6K. (Case 3; I–L) Spots with hormonal therapy showing moderate-to-strong (++) staining of phospho-PKC and phospho-S6K. Original magnification, x200.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Growing evidence suggests that AR plays an essential role in transition of androgen-independent cell proliferation in prostate cancer (3). Hence, we first examined the protein levels of AR in both AILNCaP cells and LNCaP cells. In accordance with previous reports (17, 18, 19, 20), we found that AR protein level was higher in AILNCaP cells than that in LNCaP cells under androgen-deprived condition. However, in our AILNCaP cells, the level of PSA mRNA induced by androgen stimulation was lower than that in LNCaP cells (17, 18, 19, 20), which was in contrast to the previous report (3). Additionally, bicalutamide did not have any significant effect on the growth of AILNCaP cells, but it could significantly reduce cell proliferation of LNCaP cells. Hence, ligand-dependent androgen hypersensitivity is not responsible for androgen-independent cell proliferation of AILNCaP cells.

It is well known that PI3K/Akt signaling pathway participates in androgen-independent growth of prostate cancer cells (38, 51, 52). About 60% of prostate cancer patients who develop metastases have tumors in which PI3K/Akt is activated as a result of PTEN gene mutation (53). LNCaP cells contain a frame-shift mutation in the PTEN gene and the PI3K/Akt signaling pathway is constitutively activated (53). Inhibiting PI3K/Akt signaling pathway by pharmacological agents induced apoptosis in LNCaP cells as described previously, suggesting that this signaling pathway is required for cell survival and growth in LNCaP cells (38, 52). In fact, several groups have generated androgen-independent LNCaP cells through the culture of those cells in the androgen-ablated condition (17, 18, 19, 20, 54). Pfeil et al. (52) reported that androgen-independent LNCaP cells were more resistant to PI3K inhibitors than parental LNCaP cells and showed higher phospho-Akt in the presence of LY294002. However, in our model, no significant activation of phospho-Akt was observed in AILNCaP cells. The expression of phospho-Akt was decreased to almost comparable level of LNCaP cells in the presence of LY294002. Shi et al. (20) also established three different androgen-independent LNCaP cells (LNCaP-cds). Similar to our cells, all three LNCaP-cds expressed higher level of AR without a new alternation and the amount of PSA induced by R1881 stimulation was significantly less than that of parental LNCaP cells. Although the authors did not examine the significance of signaling pathway activation in their progression to androgen-independent cell proliferation, all of LNCaP-cds expressed higher level of phospho-Akt in contrast to AILNCaP cells (20). So, mechanisms accounted for androgen-independent cell proliferation of AILNCaP cells might be different from the ones previously reported and characterized. Because Unni et al. (39) have reported that constitutive activation of Erk1/2 signaling through Src activation might play some role for transition of LNCaP cells to androgen independence, we first examined the phosphorylated form of Erk1/2 and p38 MAPK during acute androgen deprivation in LNCaP cells and compared their expression with AILNCaP cells under the same condition. Although there was no significant difference in the phosphorylated p38 MAPK between these cell lines, expression of phosphorylated Erk1/2 was significantly elevated in AILNCaP cells. However, inhibition of Erk activity with PD98059 had no significant effect on the cell proliferation in AILNCaP cells. In addition, we showed that treatment with a myristoylated PKC pseudosubstrate peptide up-regulated expression of phosphorylated Erk, whereas it impeded cell proliferation in AILNCaP cells (see the supplemental figure published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Hence, activation of Erk1/2 observed in AILNCaP cells and in LNCaP cells under androgen deprivation may be due to a compensatory mechanism to alleviate adverse effects of various cell stresses (55). Moreover, Ravi et al. (56) reported that activation of ras/raf/MAPK pathway in LNCaP cells with an inducible c-raf-1 expression plasmid caused growth suppression in these cells. So, the activation of the MAPK signaling pathway does not always induce cell proliferation in LNCaP cells.

In the present report, we found for the first time that activation of the mTOR/S6 kinase pathway was regulated by androgen in LNCaP cells, whereas this pathway was activated constitutively in AILNCaP cells under androgen-depleted conditions. Treatment with rapamycin partly reduced progression from G1 to S phase in LNCaP cells cultured with normal serum and in androgen-independent AILNCaP cells under androgen deprivation. Moreover, androgen stimulation after androgen deprivation activated the mTOR/S6 kinase pathway in the androgen-dependent LNCaP cells. These observations suggest that mTOR/S6 kinase pathway was activated by stimulation of androgen in LNCaP cells and constitutive activation of pathway was related to androgen-independent cell proliferation in AILNCaP cells. Recent studies by Ghosh et al. (57) reported that mTOR-S6 kinase activation is important for cell proliferation in androgen-independent prostate cancer cells. They also showed that activity of S6 kinase was higher in C4–2 cells in comparison to LNCaP cells in response to growth factor stimulation. Under normal cell growth condition, our results demonstrated that there was no significant difference in the S6 kinase activity between LNCaP and AILNCaP cells. Furthermore, they showed that rapamycin inhibited cell proliferation of C4–2 cells but not of LNCaP cells, which was different from our results. This might be due to difference in some experimental conditions. For instance, our treatment with rapamycin was extended for 72 h before harvesting the cells for analysis in cell cycle distribution, whereas their treatment with rapamycin was for 48 h.

To clarify the roles of androgen stimulation in activating mTOR/S6K pathway, we investigate the activities of various known upstream signaling pathways and demonstrate that activation of PKC is responsible for mTOR/S6K activation in both cells. Androgen deprivation reduced the activity of PKC in LNCaP cells, whereas androgen stimulation partially restored this activity. On the other hand, PKC was activated in AILNCaP cells and these cells showed twice as much PKC kinase activity than that of LNCaP cells in androgen-deprived condition. To demonstrate the requirement for higher PKC kinase activity for androgen-independent cell proliferation and S6K activation in AILNCaP, we examined the effect of a specific inhibitor of PKC, a myristoylated PKC pseudosubstrate peptide on these cells. Treatment with 20 µM myristoylated PKC psuedosubstrate peptide induced 20% decrease in PKC kinase activity (data not shown) and this decrease resulted in 50% reduction of cell population in S phase, 40% reduction of phospho-S6K expression, and inducing apoptosis in AILNCaP cells. These results also suggested that twice as much difference of endogenous PKC kinase activity between AILNCaP and LNCaP cells in androgen-deprived condition was significant. This result is consistent with our recent results in that 2-fold difference of endogenous PKC kinase activity was sufficient to influence the conformation of AP-1 family protein such as JunB in renal cell carcinoma cell lines (58). Inhibition of PKC kinase activity also reduced phospho-S6K expression (Fig. 7A) and cell proliferation (data not shown), and induced apoptosis in AR-negative prostate cancer cell lines, PC3 and DU145. So, this activity is also required for cell proliferation of these cells. As for the reasons why mere androgen stimulation partially restored S6K and PKC activity in LNCaP cells, we speculate that the possibility that other steroids ablated in CSFBS may also participate in the activation of these kinases. The precise mechanism for this is currently under our investigation.

As for functional relationship between PKC and S6K, the association of transfected PKC and S6K was observed in LNCaP, and this association was enhanced by androgen stimulation in LNCaP cell. We also revealed that the inhibition of endogenous PKC activity did induce the reduction of endogenous phosphorylation of S6K. Although the association of endogenous protein was not confirmed, these results indicate the possibility that PKC can associate with S6K in LNCaP cells in similar ways that was previously reported in HEK 293 cells (29) and androgen stimulation enhanced this association in LNCaP cells. Because the amount of S6K protein decreased under androgen depleted conditions, there is also a possibility that expression of S6K is regulated by PKC in mRNA or protein level (59). The precise roles in LNCaP cells remained to be clarified.

In LNCaP cells, 0.1 nM of R1881 stimulation activated PKC/S6K signaling pathway, whereas mere supplementation of R1881 with charcoal-stripped serum only modestly induced cell proliferation. Although 10 nM of R1881 stimulation activated PKC/S6K, it did not induce cell proliferation effectively in LNCaP cells. This discrepancy suggests that other signaling pathways also control cell proliferation of LNCaP cells (60, 61).

Treatment with the specific AR inhibitor, bicalutamide, together with R1881 attenuated PKC and S6K phosphorylation in LNCaP cells, indicating that the androgen/androgen-receptor complex participated in this signaling activation. Our data suggested a few possible mechanisms of androgen action to activate PKC: 1) androgen stimulation regulates PKC phosphorylation, probably through another PKC kinase; 2) androgen stimulation stabilizes PKC by regulating scaffold proteins such as molecular chaperones, or by inhibiting degradation pathways; and 3) androgen stimulation regulates a phosphatase that inactivates PKC. The precise mechanisms remained to be clarified.

Interestingly, recent evidence suggests that PKC is involved in estradiol-activated signaling pathways regulated by a classic steroid receptor without exerting a transcriptional effect in a human breast cancer cell line, MCF-7 (62). Thus, based on this previous report, important issues are raised whether androgen/AR complex participates in this signaling activation through genomic or nongenomic action (63, 64). Castoria et al. (62) revealed that stimulation with estradiol for 3 min activates PKC in MCF-7 cells and described that this nongenomic action of steroid receptors facilitates the Src-dependent Ras activation through PKC. The Src-dependent Ras activation also occurred by 2- to 5-min androgen stimulation in LNCaP cells (39, 65). In our model, R1881 treatment did not induce any apparent increase either in PKC (T410) or S6K (T389) phosphorylation within 6 h, whereas it significantly induced an increase in phosphorylation of both PKC and S6K after 24 h (data not shown). The induction of PSA was observed after 6 h at 10 nM of R1881 (data not shown). These results implied that slow receptor transcriptional activity rather than rapid signaling activation participates in PKC activation in LNCaP cells. However, these results do not exclude the possibilities of nongenomic action of AR definitively and they remain to be clarified.

In conclusion, we demonstrate for the first time that the androgen/androgen-receptor complex activates the mTOR/S6K pathways through PKC in LNCaP cells and constitutive activation of this pathway is related to transition of LNCaP cells to androgen-independent cell proliferation. We also show that both S6K and PKC are activated in considerable numbers of hormone-naive prostate cancer cells in vivo, and their activation is correlated with each other. Furthermore, activation of both kinases positively correlated with the expression of Ki67, which is a good indicator of cell proliferation (49), supporting the in vitro results. As for hormone-treated prostate cancer specimens, we analyzed residual viable cancer cells in the specimen. So, most of them might be a transition state in the continuum between hormone-naive prostate cancer and full-blown hormone-refractory cancer cells (50). Interestingly, 86% of spots were positively stained with both phospho-PKC and phosphor-S6K antibodies. We did not exclude the possibilities that some of these cells acquired hypersensitivity to androgen, and the number of hormone-treated prostate cancer specimen is too small to evaluate the significance of activation of PKC/S6K pathway definitively. However, these results implied the possibility that activation PKC-mTOR/p70 S6 kinase pathway may be associated with transition of hormone-naive prostate cancer cells to androgen-independent growth or survival also in vivo.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Antibodies and Reagents
Anti-phospho-Akt (S473), anti-phospho-p70S6K (T389), anti-phospho-p70S6K (S371), anti-phospho-S6 (S235/236), anti-phospho-p44/42 MAPK (T202/Y204), anti-phospho-p38 MAPK (T180/Y182), anti-phospho-PDK1 (S241), anti-phospho-TSC2 (T1426), anti-Akt, anti-S6, anti-p44/42 MAPK, and anti-p38 MAPK antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-PDK1, anti-AR, anti-PSA, anti-TSC2, anti-phospho-PKC (T410), anti-c-myc, anti-PKC and anti-S6K were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cyclin D1 was obtained from Novocastra (Newcastle, UK). Anti-ß-actin was purchased from Abcam (Cambridge, UK), and anti-HA, from Covance (Berkeley, CA). LY294002, rapamycin, PD98059 and myristoylated PKC pseudosubstrate inhibitor were purchased from Calbiochem (San Diego, CA). Wortmannin and hydroxyflutamide were obtained from Sigma (St. Louis, MO), and bicalutamide, from Toronto Research Chemicals (Toronto, Ontario, Canada). Hoechst 33342 was obtained from Wako (Kumamoto, Japan). R1881 (methyltrienolone) was purchased from DuPont Merck Pharmaceutical (Boston, MA).

Cell Culture
The prostate cancer cell lines LNCaP, PC3, DU145, and HEK 293 were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured routinely in RPMI (Invitrogen, Carlsbad, CA) or DMEM (Invitrogen) supplemented with 10% FBS at 37 C in incubators with humidified air and 5% carbon dioxide. The subline AILNCaP was established by maintaining LNCaP cells in phenol red-free RPMI (Invitrogen) supplemented with 10% CSFBS (Hyclone, Logan, UT), with a change of this steroid-free medium every 3–4 d over 3 months as described previously (38, 59). Although more than 99% of cells underwent apoptosis during 3 months of cell culture in CSFBS, the remaining new cell line, AILNCaP, begun to grow after this 3-month period and was maintained in phenol red-free RPMI (Invitrogen) supplemented with 10% CSFBS being passaged at 70% confluence by trypsinization, for another 6 months.

Flow Cytometry
Control and treated cells were harvested by 1 ml of 0.05% trypsin-EDTA for 3 min at 37 C to detach them from the plastic surface. Cells were centrifuged, washed in PBS, and then fixed by slow addition of 3 ml of ice-cold 70% ethanol with mildly shaking; they then were stored at 4 C until use. On the day of cycle analysis, the cells were centrifuged, washed in PBS, resuspended in 1 ml per 10⁶ cells of PBS containing 100 µg/ml RNase A (QIAGEN, Hilden, Germany) and 0.25 µg/ml of 7-amino-actinomycin D (BD Biosciencess, San Diego, CA), and incubated at 37 C for 30 min. To determine DNA content, at least 10,000 cells were analyzed with a FACSCalibur flow cytometer using CellQuest software (BD Biosciences).

Quantitative RT-PCR
Total cellular RNA was isolated with RNeasy Mini Kit (QIAGEN), and cDNA was synthesized from 2 µg of total RNAs with random primers using First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instruction. PCR was performed by SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA) as described using the relative standard curve method (66). The increase in fluorescence of the SYBR green dye was moniterd using GeneAmp 5700 sequence detection system (Applied Biosystems). All of the PCRs were performed in triplicate. The values were normalized to the amounts of TATA-binding protein. The sequences of primers used for PCR analyses are as follows: PSA, 5'-GGAAATGACCAGGCCAAGAC-3' (sense) and 5'-CAACCCTGGACCTCACACCTA-3' (antisense), TATA-binding protein (TATABP), 5'-GAATATAATCCCAAGCGGTTTG-3' (sense) and 5'-ACTTCACATCACAGCTCCCC-3' (antisense). Conventional PCR was conducted with the following profile: initial heating to 95 C for 10 min followed by 37 PCR cycles of denaturing at 95 C for 45 sec, annealing at 60 C for 45 sec, and extension at 72 C for 45 sec for both PSA and TATABP. The amplified products were visualized on 1.8% agarose gels.

Cell Lysis and Immunoblotting
After washing with ice-cold PBS, cells were harvested in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM sodium chloride, 2 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 50 mM sodium fluoride, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride supplemented with protease cocktail inhibitors (Complete Mini; Roche, Mannheim, Germany). Whole-cell extracts were centrifuged at 13,000 x g at 4 C for 20 min. Total cellular protein concentrations were determined by using a protein assay reagent (Bio-Rad, Richmond, CA). Lysates were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were immunoblotted with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies, and developed for reading by enhanced chemiluminescence (Amersham Pharmacia Biotech). Densitometric analysis was performed by Image J software (National Institutes of Health, Bethesda, MD). All blots were stained with Ponceau S to confirm equal protein loading.

Transfection and Immunoprecipitation
Transfection was performed in DMEM or RPMI with or without 10% FBS using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Cells were lysed in lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% (vol/vol) Nonidet P-40, 5 mM EDTA] containing 2 mM orthovanadate, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and protease cocktail inhibitor, and immunoprecipitated with the antibody indicated and protein G-Sepharose beads (Amersham, Buckinghamshire, UK). Immune complexes were subjected to SDS-PAGE and Western blotting.

Expression Constructs
PKC-myc was generated by subcloning wild-type human PKC into the KpnI and EcoRI sites of the mammalian expression vector pcDNA3.1/myc-His A (Invitrogen). All constructs were amplified by PCR, and DNA sequences were verified using ABI PRISM 310 genetic analyzer. HA-p70S6K wild-type was kindly provided by Dr. John Blenis (Department of Cell Biology, Harvard Medical School, Boston, MA).

Immunocomplex Kinase Assay
Cells in the growth phase were washed with PBS and lysed in lysis buffer [50 mM Tris-Hcl (pH 7.4), 150 mM NaCl, 0.5% (vol/vol) Nonidet P-40, 5 mM EDTA] containing 2 mM orthovanadate, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and protease cocktail inhibitor. Activity of S6 kinase was determined using a S6 kinase assay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer’s instructions with some modification. Briefly, 10 µl of assay dilution buffer [ADB: 20 mM 3[N-morpholino]propanesulfonic acid (pH 7.2), 25 mM ß-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mM dithiothreitol], 10 µl of substrate cocktail [250 µM substrate peptide (AKRRRLSSLRA) in ADB], 10 µl of inhibitor cocktail, 10 µl of the [-³²P] ATP mixture (magnesium/ATP cocktail including 10 µCi of [-³²P] ATP) and immunoprecipitate with S6 kinase polyclonal antibody (Santa Cruz) were mixed and incubated for 10 min at 37 C. For assay of PKC activity, lysates were prepared similarly, immunoprecipitated with PKC polylclonal antibody, and then incubated for 20 min at 30 C in 50 µl of kinase assay mixture as described previously (35). In all kinase reactions, ³²P incorporation into substrates was measured by liquid scintillation counting.

Prostate Cancer Tissue Microarray and Immunohistochemistry
Prostate cancer tissues evaluated were derived from radical prostatectomy specimens of 79 localized prostate cancer patients at Kyoto University Hospital. Using these specimen TMAs were constructed as previously described (49). Standard indirect immunoperoxidase procedures using monoclonal and polyclonal antibodies were applied to detect AR (1:100, 2F12 Novocastra), Ki67 (1:100, MIB-1, DAKO, Kyoto, Japan), phospho-p70S6K (T389) (1:50; Cell Signaling Technology), and phospho-PKC (T410) (1:100; Santa Cruz). Adjacent sections within 15 µM of TMAs were used for the analysis. Available cancer spots were 213 spots. Immunopositivity of phospho-p70S6K and phospho-PKC was graded as (–) (no staining), (+) (weak immunostaining involving less than 50%), (++) (moderate-to-strong immunostaining involving more than 50%) by two of the authors (T. I. and Y.S.), independently. The Ki67 labeling index was determined as described previously (49). All the patients involved in this study provided informed consent.

Statistical analyses
Data are presented as mean ± SD. Means were considered as statistically different, i.e. P < 0.05, which was determined by one-way ANOVA and the least significant multiple comparison method. Spearman’s rank order correlation analysis was used to analyze the statistical significance of the correlations among phospho-S6K and phospho-PKC expression in cancer spots.

	ACKNOWLEDGMENTS

 
We thank Dr. John Blenis (Department of Cell Biology, Harvard Medical School, Boston, MA) for supplying pRK7 HAS6K-1. We also thank all members of Cancer Research Course in Graduated Courses for Integrated Research Training, Kyoto University Faculty of Medicine, the Ogawa’s lab, and Dr. Tomomi Yamada (Division of Medical Informatics, Kyusyu University Hospital) for helpful discussion. We thank the skillful technical assistance of Tomoko Matsushita and Chie Hagihara (Department of Urology, Kyoto University).

	FOOTNOTES

 
This work was supported by a Grant-in-Aid from Ministry of Education, Culture, Sports, Science and Technology, Japan, Yamaguchi Endocrine Research Association, the Japanese Foundation for Prostate Research, Organon Urology Academia, and Formation for Genomic Analysis of Disease Model Animals with Multiple Genetic (Center of Excellence program), Ministry of Education, Culture, Sports, Science and Technology, Japan.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 24, 2006

Abbreviations: aPKC, Atypical PKC; AR, androgen receptor; CSFBS, charcoal-stripped fetal bovine serum; HEK, human embryonic kidney; mTOR, mammalian target of rapamycin; PDK-1, 3-phosphoinositide-dependent protein kinase 1; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PSA, prostate-specific antigen; TMA, tissue microarray.

Received for publication January 19, 2006. Accepted for publication August 14, 2006.

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