Chemotherapy of prostate cancer targets androgen receptor (AR) by androgen ablation or antiandrogens, but unfortunately, it is not curative. Our attack on prostate cancer envisions the proteolytic elimination of AR, which requires a fuller understanding of AR turnover. We showed previously that calmodulin (CaM) binds to AR with important consequences for AR stability and function. To examine the involvement of Ca2+/CaM in the proteolytic breakdown of AR, we analyzed LNCaP cell extracts that bind to a CaM affinity column for the presence of low molecular weight forms of AR (intact AR size, ∼114 kDa). Using an antibody directed against the NH2-terminal domain (ATD) of AR on Western blots, we identified ∼76-kDa, ∼50-kDa, and 34/31-kDa polypeptides in eluates of CaM affinity columns, suggesting the presence of CaM-binding sites within the 31/34-kDa ATD of AR. Under cell-free conditions in the presence of phenylmethylsulfonyl fluoride, AR underwent Ca2+-dependent degradation. AR degradation was inhibited by N-acetyl-leu-leu-norleu, an inhibitor of thiol proteases, suggesting the involvement of calpain. In intact cells, AR breakdown was accelerated by raising intracellular Ca2+ using calcimycin, and increased AR breakdown was reversed with the cell-permeable Ca2+ chelator bis-(O-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetra-(acetoxymethyl)-ester. In CaM affinity chromatography studies, the Ca2+-dependent protease calpain was bound to and eluted from the CaM-agarose column along with AR. Caspase-3, which plays a role in AR turnover under stress conditions, did not bind to the CaM column and was present in the proenzyme form. Similarly, AR immunoprecipitates prepared from whole-cell extracts of exponentially growing LNCaP cells contained both calpain and calpastatin. Nuclear levels of calpain and calpastatin (its endogenous inhibitor) changed in a reciprocal fashion as synchronized LNCaP cells progressed from G1 to S phase. These reciprocal changes correlated with changes in AR level, which increased in late G1 phase and decreased as S phase progressed. Taken together, these observations suggest potential involvement of AR-bound CaM in calcium-controlled, calpain-mediated breakdown of AR in prostate cancer cells. (Cancer Res 2006; 66(24): 11754-62)

Adenocarcinoma of the prostate is the most frequently diagnosed cancer and second leading cause of cancer deaths in American men (1). Although androgen ablation is the most common therapy for disseminated prostate cancer, it is palliative in nature and most patients eventually succumb to hormone-refractory disease resistant to chemotherapy. Whether normal or mutated, androgen receptor (AR) is required for growth in both androgen-sensitive and androgen-insensitive prostate cancer (2). Therefore, it is of paramount importance to dissect the various ways in which AR is regulated to not simply inactivate but to physically eliminate AR (3). This requires understanding the cellular mechanisms of AR breakdown.

Numerous studies have examined the proteolytic destruction of AR, and our knowledge can at present be divided into three areas. First, AR is degraded to small (≤10 residues) peptides by the threonine proteases of the ubiquitin-proteasome system (49). Second, AR can be cleaved by serine proteases (1016). These studies established that, structurally, AR consists of at least three domains, the ligand-binding domain (LBD), the DNA-binding domain (DBD), and the NH2-terminal domain (ATD), separated by protease-sensitive linker regions. Serine proteases cleave AR to yield a 34-kDa COOH-terminal LBD (12, 17) and the remaining ∼76 kDa of AR comprising the ATD and DBD. Other studies (11) established that proteolysis can cleave AR into two fragments: one of ∼50 kDa containing both the LBD and the DBD and another of approximately the same size, presumably the ATD (13). Third, the caspase family of thiol proteases is capable of cleaving AR, particularly those forms of AR with expanded polyglutamine repeats (1822). To date, the cleavage of AR by the Ca2+-activated, thiol protease calpain has not been described.

The amount, localization, physiologic functions, and degradation of AR are influenced by the following factors. First, AR is transcribed, translated (23, 24), and post-translationally phosphorylated (25, 26). Androgen binds to the LBD, inducing conformational change critical to the transcription factor activity of AR as well as resistance to degradation (17, 27). Following androgen binding, AR is ready to act as a transcription factor (23), as the DBD binds to the androgen response element (ARE). The activity of the DNA-bound complex is governed by binding of coactivator and corepressor proteins to the LBD and the ATD. Finally, AR activity is also controlled by AR-calmodulin (CaM) interaction (28). It is the effect of AR interaction with CaM on the breakdown of AR that we examine herein.

Extending our observation that CaM binds to AR directly (28), we now map the CaM-binding site(s) to the ATD of AR. We show for the first time that Ca2+ stimulates AR breakdown and that CaM bound to AR may play an important role in regulating AR levels by engaging calpain/calpastatin in cleavage of AR under physiologic conditions. Other pathways involving caspase-3 may also play a role in AR breakdown under stress conditions. These observations raise the possibility that anti-CaM drugs and calpastatin inhibitors can be used in the treatment of prostate cancer, particularly when the disease is metastatic and hormone refractory.

Cell culture. LNCaP cells (obtained from the American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 (Life Technologies, Grand Island, NY) containing 10 nmol/L testosterone (Sigma Chemical Co., St. Louis, MO) supplemented with 10% FCS (Atlanta Biologicals, Lawrenceville, GA), 2.5 mmol/L glutamine, 100 μg/mL streptomycin, and 100 units/mL penicillin. Cells were maintained in a humidified incubator with 5% CO2 and 95% air at 37°C. PBS (pH 7.4) without magnesium or calcium (10× concentrate), glutamine, and antibiotics were obtained from Life Technologies.

Synchronization of LNCaP cells by isoleucine deprivation. Isoleucine deprivation was done by our previously published method (29). The cells were grown to 60% to 70% confluence, and then the medium was replaced with isoleucine-free RPMI 1640 supplemented with 6% dialyzed FCS (Life Technologies), 2.5 mmol/L glutamine, 100 μg/mL streptomycin, 100 units/mL penicillin, and 10 nmol/L testosterone. Cells were maintained in isoleucine-free medium for 36 hours in an incubator as above. Cells were released from isoleucine block by replacing the medium with the corresponding complete medium containing 10% FCS.

Preparation of nuclear and cytoplasmic fractions. LNCaP cells were subjected to nuclear and cytoplasmic fractionation by slight modification of the method of Subramanyam et al. (30). All chemicals were obtained from Sigma-Aldrich (St. Louis, MO), except where otherwise noted. After harvest, cells were pelleted by centrifugation for 10 minutes at 4°C at 2,000 rpm in a Sorvall RT7 centrifuge and resuspended in buffer A [0.16 mol/L sucrose, 50 mmol/L Tris-HCl (pH 7.6), 25 mmol/L KCl, 10 mmol/L MgCl2, 70 mmol/L HEPES (pH 7.6), 0.025 mmol/L CaCl2, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 2 mmol/L DTT, 1 mmol/L EDTA] at a density of 2 × 107/mL. The cell suspension was then homogenized in a top-driven Wheaton homogenizer (Wheaton Co., Wheaton, IL) until ∼90% of the cells were stained with trypan blue dye (usually three strokes at a rotation setting of 3). The cytosolic supernatant was then separated from the nuclear pellet by centrifugation at 4°C in a Beckman Microfuge B at 6,000 rpm for 5 minutes (Beckman Instruments, Palo Alto, CA). A volume of buffer A equal to that used for homogenization was added to the microfuge tube, and the nuclei were then subjected twice to 30 pulses of sonication with a Branson Sonifier 250 (Branson Co., Danbury, CT) set at an output control of 2 and a duty cycle of 20, with intermittent cooling on ice. The sonicated nuclear extract was cleared by centrifugation in the microfuge as described above. Protein concentration in cytosolic and nuclear extracts was assessed with the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) using the Bradford reagent in 96-well plates and read using an EL340 Micro Colorimeter (BioTek Instruments, Winooski, VT).

Whole-cell extracts were prepared essentially as described by Cifuentes et al. (28). Briefly, LNCaP cells were suspended at a density of 2 × 107/mL in buffer A and sonicated and the extracts were clarified as above.

UVB irradiation. UV irradiation was administered (31) using a single FS40 lamp (National Biological, Twinsburg, OH). FS40 lamps produce 0.5% UVC (260–280 nm), 62% UVB (280–320 nm), and 38% UVA (320–400 nm) with a peak emission at 313 nm. The average irradiance of the source was approximately 1.58 × 10−4 W at 17-cm distance as measured by an IL1700 Research Radiometer (International Light, Newburyport, MA) with a UVB-1 filter (#25628) and SED 240 detector. LNCaP cells were grown to 70% confluence in P100 plates. Immediately before UV radiation treatment, the medium was removed from each culture, the cells were washed once with PBS, and fresh PBS was added. Cultures were exposed to one of two doses of UV radiation (300 J/m2 for 40 seconds or 600 J/m2 for 80 seconds) under sterile conditions with the cover of the culture dish off. Following UV exposure, the PBS was removed and replaced with serum-containing culture medium and cultured for an additional 12 or 24 hours. Control cultures were identically treated but were not UV irradiated. The cells were then harvested by pipetting the culture medium up and down on the cells with a Pasteur pipette until all cells were detached. The cells were washed once in PBS/EDTA before sonicating to prepare cell extracts as above.

Treatment of cells with Ca2+ ionophore and intracellular Ca2+ chelator. Subconfluent, P150 plates of exponentially growing cultures of LNCaP cells were treated with the Ca2+ ionophore A23187 (calcimycin) or the cell-permeable calcium chelator 1,2-bis-(O-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetra-(acetoxymethyl)-ester (AM-BAPTA; Molecular Probes, Eugene, OR) for 24 hours. BAPTA or control vehicle was added to cultures and incubated at 37°C. Thirty minutes later, A23187 was added and cells were incubated for 24 hours. The cells were harvested, as above under UV treatment, by pipetting the culture medium up and down to detach the cells followed by washing and sonication to prepare the extracts.

Western blot analysis. Samples were dissolved in PAGE loading buffer (Bio-Rad, Richmond, CA) and subjected to denaturing 10% or 12% SDS/PAGE and then transferred to nitrocellulose membranes. Individual membranes were probed with rabbit polyclonal antibody (pAb) against the 20 NH2-terminal residues (N20) or murine monoclonal antibody (mAb) against residues 301 to 317 (mAb 441) of human AR (Santa Cruz Biotechnology, Santa Cruz, CA). Other primary antibodies used were mAb against the following: CaM (Upstate Cell Signaling Solutions, Lake Placid, NY); caspase-3 (Santa Cruz Biotechnology); the COOH-terminal domain of poly(ADP-ribose) polymerase (PARP), which reacts with both the intact 112-kDa and the clipped 85-kDa forms of PARP (Santa Cruz Biotechnology); μ-calpain large subunit and calpastatin (Chemicon International, Temecula, CA); and rabbit mAb to cleaved caspase-3 (Cell Signaling Technology, Beverly, MA). Immunoreactive bands were developed by using horseradish peroxidase–conjugated secondary antibodies and SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL) and visualized by using X-ray film.

CaM-agarose affinity chromatography. The CaM affinity chromatography procedure was essentially as described by Cifuentes et al. (28). Briefly, nuclear (∼2.5 mg protein) and cytoplasmic (∼7.5 mg protein) lysates of LNCaP cells prepared as described above were applied to 2 mL CaM-agarose affinity columns (Sigma Chemical) poured in 25-cm Econocolumns (Bio-Rad). Nonadherent proteins were washed from the column with buffer B [20 mmol/L Tris (pH 7.4), 4 mmol/L MgCl2, 2 mmol/L CaCl2, 10 mmol/L KCl, 1 mmol/L PMSF] until A280 returned to the baseline. This nonadherent fraction was called the drop thru. Proteins retained on the column were then eluted with modified buffer B [20 mmol/L Tris (pH 7.4), 4 mmol/L MgCl2, 10 mmol/L EGTA, 10 mmol/L KCl, 1 mmol/L PMSF] in which the Ca2+ chelator EGTA was substituted for Ca2+. All chromatography procedures, including cell lysate application, washing, and elution, were carried out without application of pressure to the gel bed other than the hydrostatic pressure of 15-cm (maximum) column of liquid above the gel. Protein in the drop thru and eluate fractions was concentrated by precipitation in ice-cold 5% trichloroacetic acid and dissolved in PAGE loading buffer.

Immunoprecipitation. AR immunoprecipitation to analyze AR-interacting proteins was done by modification of the method of Cifuentes et al. (28). Cell extracts were diluted 10-fold in 250 mmol/L NaCl and 50 mmol/L Tris (pH 7.4; dilution buffer) and incubated overnight with 4 μg/mL of N20 or 441 anti-AR antibody at 4°C. The immune complexes were then adsorbed to protein A/G agarose immunoadsorbent (Pierce) by incubation for 1 hour at ambient temperature with gentle agitation. The adsorbed complexes were washed twice in dilution buffer by centrifugation at 4°C and eluted with PAGE loading buffer. Mock immunoprecipitates were obtained by diluting and incubating the extracts without AR antibody. The mixture was then incubated with immunoadsorbent and eluted as above.

CaM-binding site in the NH2-terminal region of AR. In LNCaP cell extracts prepared from cells harvested by trypsinization, there was a noticeable breakdown of AR to fragments with approximate molecular weights of ∼50 and 31/34 kDa. Like intact AR, each of these AR fragments was detected on Western blots using affinity-purified pAbs raised against the most NH2-terminal 20 amino acids of human AR (pAb N20, Santa Cruz Biotechnology; Fig. 1). Therefore, the AR fragments detected by pAb N20 are referred to as AR ATD fragments. Following up on our observation that AR in LNCaP cell extracts binds to CaM (28), we attempted to map the CaM-binding region on AR by testing whether the ∼50-kDa or 31/34-kDa AR ATD fragments retain CaM-binding ability. CaM-agarose affinity chromatography of lysate from trypsinized LNCaP cells showed strong binding of both the ∼50-kDa and 31/34-kDa AR ATD fragments to CaM. Although Ponceau S stainable protein could be detected equally well in both the “drop thru” and EGTA eluate fractions of a CaM-agarose affinity column (data not shown), the pAb N20 immunoreactive bands of AR and AR ATD fragments could only be detected in the EGTA eluate (Fig. 1, lane 2). In addition, we observed that, whereas the pAb N20 detected all ATD fragments of AR, mouse mAb (32) raised against amino acids 301 to 317 of the AR (mAb 441, Santa Cruz Biotechnology) detected intact AR and the ∼50-kDa ATD fragment but not the 31/34-kDa ATD fragment (data not shown). These observations together suggest that the CaM-binding site lies within the 31/34-kDa NH2-terminal region of AR and that the 31/34-kDa fragment lacks the 301 to 317 epitope.

Figure 1.

AR and its breakdown products in LNCaP cell lysate and CaM-agarose affinity column fractions. An ∼1 mL volume of cell lysate of 2 × 107 LNCaP cells (harvested by trypsinization) was applied to 2 mL bed volume columns of CaM-agarose in affinity chromatography buffer containing 2 mmol/L Ca2+ and 1 mmol/L PMSF. After nonadherent material (drop thru) washed through, CaM-binding materials were eluted with buffer lacking Ca2+ but containing 10 mmol/L EGTA (EGTA Eluate). Each lane was loaded with ∼20 μg protein and respectively contained the following: crude cell lysate (lane 1), material not binding to CaM, drop thru (lane 2), and material eluted from CaM with EGTA (lane 3). After PAGE and transfer, the blots were stained with the pAb N20 and developed with anti-rabbit conjugated antibody as described in Materials and Methods. Data are representative of two experiments, each comprising four chromatographic runs.

Figure 1.

AR and its breakdown products in LNCaP cell lysate and CaM-agarose affinity column fractions. An ∼1 mL volume of cell lysate of 2 × 107 LNCaP cells (harvested by trypsinization) was applied to 2 mL bed volume columns of CaM-agarose in affinity chromatography buffer containing 2 mmol/L Ca2+ and 1 mmol/L PMSF. After nonadherent material (drop thru) washed through, CaM-binding materials were eluted with buffer lacking Ca2+ but containing 10 mmol/L EGTA (EGTA Eluate). Each lane was loaded with ∼20 μg protein and respectively contained the following: crude cell lysate (lane 1), material not binding to CaM, drop thru (lane 2), and material eluted from CaM with EGTA (lane 3). After PAGE and transfer, the blots were stained with the pAb N20 and developed with anti-rabbit conjugated antibody as described in Materials and Methods. Data are representative of two experiments, each comprising four chromatographic runs.

Close modal

AR cleavage is accentuated in LNCaP cell extracts subjected to CaM affinity column. Quantitative analysis of the relative distribution of pAb N20 immunoreactive bands of intact and ATD fragments of AR in cell lysates versus those enriched in the EGTA eluate of a CaM affinity column suggested that AR in LNCaP cell lysates may have undergone additional proteolytic breakdown during the course of affinity chromatography despite the presence of PMSF in the buffer. Densitometry of pAb N20 immunoreactive bands in cell lysate showed an approximate distribution of intact AR and the ∼50-kDa and 31/34-kDa ATD fragments to be 38%, 36%, and 26%, respectively (Table 1). By comparison, the same cell lysates, when subjected to CaM affinity chromatography, yielded EGTA eluate that contained pAb N20 immunoreactive bands of intact AR and the ∼50-kDa and 31/34-kDa ATD fragments with a density distribution of 9%, 43%, and 43%, respectively (Table 1). Thus, there was a significant decrease of intact AR and an increase of 50-kDa and 31/34-kDa ATD fragments in the EGTA eluate fraction compared with that in the cell lysate before CaM affinity chromatography.

Table 1.

Densitometric analysis of the Western blot in Fig. 1 to determine the degree of breakdown of AR in the initial sample and in the EGTA eluate

Integrated densitometric gray-scale units* (% of total in lane)
Form of ARCell lysate, n (%)Drop thruEGTA eluate, n (%)
Intact AR 114,249 (38) 8,167 90,001 (9) 
∼50-kDa fragment 107,436 (36)  432,440 (43) 
31/34-kDa fragment 78,006 (26)  428,377 (43) 
Integrated densitometric gray-scale units* (% of total in lane)
Form of ARCell lysate, n (%)Drop thruEGTA eluate, n (%)
Intact AR 114,249 (38) 8,167 90,001 (9) 
∼50-kDa fragment 107,436 (36)  432,440 (43) 
31/34-kDa fragment 78,006 (26)  428,377 (43) 
*

On an arbitrary scale of 0 to 256 density units above background per pixel determined using the Stratagene (La Jolla, CA) Eagle Eye II system.

Ca2+ stimulates AR breakdown in LNCaP cell extracts. The cleavage of AR on chromatography on CaM affinity columns observed above occurred in the presence of 1 mmol/L PMSF, suggesting that AR proteolysis was caused by an enzyme that was not a serine esterase. Given that the loading buffer for applying extracts to the CaM affinity column contained 2 mmol/L Ca2+, we sought to determine if Ca2+ was activating a proteolytic factor in the cell extracts. Therefore, LNCaP nuclear extracts (prepared from cells harvested by scraping rather than trypsinization) were incubated for varying times in the presence of increasing concentrations of Ca2+ before being separated by PAGE and analyzed by Western blots using pAb N20. Figure 2A illustrates that AR proteolysis proceeded even in the presence of the low level of Ca2+ that was present in the buffer used for the preparation of nuclear extract at 4°C. Increasing Ca2+ concentration to the millimolar range caused complete breakdown of AR into lower molecular weight fragments over a 48-hour incubation at 4°C. Similar effects were seen with cytosolic extracts at 4°C and with both nuclear and cytosolic extracts at 23°C, albeit at a more rapid rate (data not shown). Thus, the enzyme responsible for production of ATD fragments from AR is Ca2+ dependent.

Figure 2.

Breakdown of AR is activated by Ca2+ and inhibited by ALLN. Nuclear extracts were prepared from exponentially growing LNCaP cells harvested by scraping. Aliquots were incubated at 4°C for 48 hours in the presence of varying (0–5 mmol/L) concentrations of Ca2+ and in the absence (A) or presence (B) of the tripeptide cysteine protease inhibitor ALLN. All reactions were terminated by addition of 35 μL of sample into 1 mL of ice-cold 5% trichloroacetic acid. PAGE gels were run with ∼7 μg protein, and Western blots were developed with either rabbit pAb to the ATD of human AR (top) or antibody to actin (bottom) to ascertain the degree of protein loading as described in Materials and Methods. Data are representative of four experiments.

Figure 2.

Breakdown of AR is activated by Ca2+ and inhibited by ALLN. Nuclear extracts were prepared from exponentially growing LNCaP cells harvested by scraping. Aliquots were incubated at 4°C for 48 hours in the presence of varying (0–5 mmol/L) concentrations of Ca2+ and in the absence (A) or presence (B) of the tripeptide cysteine protease inhibitor ALLN. All reactions were terminated by addition of 35 μL of sample into 1 mL of ice-cold 5% trichloroacetic acid. PAGE gels were run with ∼7 μg protein, and Western blots were developed with either rabbit pAb to the ATD of human AR (top) or antibody to actin (bottom) to ascertain the degree of protein loading as described in Materials and Methods. Data are representative of four experiments.

Close modal

Ca2+-stimulated breakdown of AR in cell extracts is attenuated by N-acetyl-leu-leu-norleu. One of the best-characterized Ca2+-activated proteases is calpain. Therefore, we tested whether the breakdown of AR into ATD fragments might be inhibited by the tripeptide analogue thiol protease inhibitor N-acetyl-leu-leu-norleu (calpain inhibitor I or ALLN). Addition of 20 μmol/L ALLN to the incubation system described above in Fig. 2A resulted in protection of AR from Ca2+-activated proteolytic cleavage into ATD fragments (Fig. 2B). A similar inhibitory effect of ALLN on Ca2+-stimulated AR breakdown was observed in cytosolic extracts incubated at 4°C and with both nuclear and cytosolic extracts incubated at 23°C (data not shown). Thus, Ca2+-stimulated AR cleavage and ALLN attenuation of Ca2+-stimulated cleavage suggest the involvement of calpain in AR cleavage.

Ca2+ ionophore stimulates and cell-permeable Ca2+ chelator suppresses breakdown of endogenous AR in LNCaP cells. Given that the breakdown of AR in vitro is accelerated by increasing the levels of Ca 2+, we investigated the effect of artificially altering intracellular levels of Ca2+. We cultured cells in the presence of the cell-permeable Ca2+ chelator acetomethyl ester form of BAPTA (AM-BAPTA) and/or in the presence of A23187, an ionophore that raises intracellular Ca2+ levels. Cells in this study were harvested by scraping rather than trypsinization. Treatment of LNCaP cells with AM-BAPTA had little effect, per se, on the level of intact AR but attenuated the formation of the ∼50-kDa and 31/34-kDa fragments of AR (Fig. 3, compare lanes 1 and 2). This is consistent with a potential role of Ca2+-dependent AR cleavage. On the other hand, treatment of LNCaP cells with the A23187 calcimycin ionophore had a striking effect. Cell rounding and detachment were observed, consistent with induction of apoptosis, although, within the 24-hour time course of the experiment, cellular integrity was unaffected as evidenced by the equal amount of actin in equal volumes of extract (Fig. 3). Relative to actin, A23187 calcimycin caused a substantial decrease in intact AR and AR fragments (Fig. 3, lane 3). This A23187-induced decrease in AR was also associated with a decrease in calpastatin, a natural inhibitor of calpain, and calpastatin breakdown products (Fig. 3). Pretreatment of LNCaP cells with AM-BAPTA partially ameliorated the effect of calcimycin ionophore (Fig. 3, lane 4).

Figure 3.

Increasing intracellular Ca2+ by ionophore increases AR breakdown, and chelation of intracellular Ca2+ decreases AR breakdown. LNCaP cells were incubated for 24 hours with the ionophore calcimycin (A23187) in the presence or absence of the intracellular Ca2+ chelator AM-BAPTA. Cells were then harvested by scraping into medium, and experimental and control (whole cell) lysates were prepared at a concentration of 2 × 107 cells/mL. PAGE was done, and the blots were analyzed with antibodies to AR (pAb N20), calpastatin, CaM, or actin as described in Materials and Methods. The experiment was repeated twice, and representative data are shown.

Figure 3.

Increasing intracellular Ca2+ by ionophore increases AR breakdown, and chelation of intracellular Ca2+ decreases AR breakdown. LNCaP cells were incubated for 24 hours with the ionophore calcimycin (A23187) in the presence or absence of the intracellular Ca2+ chelator AM-BAPTA. Cells were then harvested by scraping into medium, and experimental and control (whole cell) lysates were prepared at a concentration of 2 × 107 cells/mL. PAGE was done, and the blots were analyzed with antibodies to AR (pAb N20), calpastatin, CaM, or actin as described in Materials and Methods. The experiment was repeated twice, and representative data are shown.

Close modal

Calpain, but not caspase-3, binds to CaM affinity columns along with AR. Cleavage of AR by calpain has not been previously reported, whereas there are numerous reports of caspase-3-mediated AR breakdown (1922). Furthermore, ALLN is not specific for calpain. Therefore, we sought further evidence for the involvement of calpain in AR degradation. LNCaP cell extracts were subjected to CaM-agarose affinity chromatography under the conditions described in Fig. 1, a procedure found to accelerate AR cleavage. The drop thru and EGTA eluate fractions were then analyzed by Western blots probed with antibodies to caspase-3, μ-calpain, and AR (Fig. 4). Caspase-3 failed to bind to CaM and was present only in the drop thru fraction (Fig. 4, lane 1). Furthermore, the molecular weight of the material immunoreactive with the antibody to caspase-3 was consistent with the presence of caspase-3 in its inactive form. No material was observed in these extracts that reacted with antibody to cleaved caspase-3 (data not shown), which is the active form of the enzyme. All of the AR bound to the CaM column and was eluted with EGTA (Fig. 4, lane 2). Calpain bound to the CaM-agarose column and was present in the EGTA eluate (Fig. 4, lane 2). Thus, the Ca2+-dependent binding of AR and calpain to CaM affinity columns is consistent with a potential role of calpain in AR cleavage.

Figure 4.

Calcium-dependent binding of AR and calpain, but not caspase-3, to CaM-agarose columns. LNCaP cells were harvested by trypsinization, extracts were subjected to CaM-agarose affinity chromatography, and the unbound flow-through protein fraction (Drop-Thru) and the protein fraction eluted with EGTA (EGTA Eluate) were subjected to Western blot analysis using antibodies to AR, μ-calpain, or caspase-3 as described in Materials and Methods. Data are from one of four chromatographic runs.

Figure 4.

Calcium-dependent binding of AR and calpain, but not caspase-3, to CaM-agarose columns. LNCaP cells were harvested by trypsinization, extracts were subjected to CaM-agarose affinity chromatography, and the unbound flow-through protein fraction (Drop-Thru) and the protein fraction eluted with EGTA (EGTA Eluate) were subjected to Western blot analysis using antibodies to AR, μ-calpain, or caspase-3 as described in Materials and Methods. Data are from one of four chromatographic runs.

Close modal

Calpain, calpastatin, and CaM, but not caspase-3, are in AR immunoprecipitates. The previous experiments suggested that calpain, but not caspase-3, was responsible for calcium-activated cleavage of AR to ATD fragments. Therefore, we sought evidence using immunoprecipitation that calpain is associated with AR in whole-cell extracts. Caspase-3, although present in crude lysates, did not coprecipitate when AR was immunoprecipitated with either mAb 441 or pAb N20 (Fig. 5), consistent with our conclusion that caspase-3 was not responsible for AR breakdown in our experiments. CaM, on the other hand, coprecipitated with AR in immunoprecipitates prepared with both antibodies (anti-AR mAb 441 and pAb N20), confirming our earlier report of AR binding to CaM (28). Calpain and calpastatin also coprecipitated with AR (Fig. 5). Coprecipitation of calpastatin with AR was more apparent with the mAb 441 than with pAb N20. We infer from this that the binding of calpastatin to AR may be disrupted when pAb N20 binds to the most NH2-terminal residues of AR. Thus, it seems that, in crude lysates at a concentration of Ca2+ that is physiologic for the intracellular milieu, AR, CaM, calpain, calpastatin, and Ca2+ form a complex.

Figure 5.

Calpain, calpastatin, and CaM, but not caspase-3, are associated with AR immunoprecipitates. Exponentially growing LNCaP cells were harvested by scraping. Whole-cell extracts (Cell Lysate) were incubated with either mAb (mAb 441) to a synthetic peptide comprising residues 301 to 317 in the middle of the ATD of AR, pAb (pAb N20) to a synthetic peptide comprising residues 1 to 20 of the ATD of AR, or immunoprecipitation buffer lacking antibody (No Ab). The mixtures were then treated with an immunoglobulin adsorbent (protein A/G mix), and the complexes were washed and then eluted with PAGE buffer. Eluates were subjected to PAGE, and Western blots were developed with antibodies to AR, calpain, calpastatin, CaM, and caspase-3 as described in Materials and Methods. The experiment was repeated thrice, and representative blots are shown.

Figure 5.

Calpain, calpastatin, and CaM, but not caspase-3, are associated with AR immunoprecipitates. Exponentially growing LNCaP cells were harvested by scraping. Whole-cell extracts (Cell Lysate) were incubated with either mAb (mAb 441) to a synthetic peptide comprising residues 301 to 317 in the middle of the ATD of AR, pAb (pAb N20) to a synthetic peptide comprising residues 1 to 20 of the ATD of AR, or immunoprecipitation buffer lacking antibody (No Ab). The mixtures were then treated with an immunoglobulin adsorbent (protein A/G mix), and the complexes were washed and then eluted with PAGE buffer. Eluates were subjected to PAGE, and Western blots were developed with antibodies to AR, calpain, calpastatin, CaM, and caspase-3 as described in Materials and Methods. The experiment was repeated thrice, and representative blots are shown.

Close modal

Changes in calpain and calpastatin levels are associated with progression of synchronized LNCaP cells from G0-G1 to S phase. Given that AR, CaM, calpain, and calpastatin seem to form a complex in the living cell and given that intracellular Ca2+ fluctuates significantly during the cell cycle (33), we examined the possibility that variation of AR levels throughout the cell cycle might be associated with concomitant changes in the levels of calpain and calpastatin. For this, we used LNCaP cells synchronized by isoleucine deprivation that progress synchronously from G0-G1 to S phase on release from isoleucine block (29). In these cells, AR levels fluctuated during the cell cycle from a low at G0-G1 (0–4 hours after release from isoleucine block) to a maximum just as S phase begins (12–16 hours after release from isoleucine block; Fig. 6). These changes were more pronounced in nuclear extracts (Fig. 6B,, top) than in cytosolic extracts (Fig. 6A , top). In both compartments, AR ATD fragments generally tended to increase as the cell cycle progressed.

Figure 6.

AR cleavage is associated with changes in calpain and calpastatin levels during the progression of synchronized LNCaP cells from G0-G1 to S phase. LNCaP cells were synchronized by isoleucine deprivation as described in Materials and Methods. Following release from isoleucine block, cells were harvested (trypsinized) either at various times corresponding to key phases of the cell cycle (0 hour, G0-G1; 4–8 hours, early G1; 12–16 hours, late G1; and 20–24 hours, S phase) or from exponentially growing cultures (Expo). Cytosol extracts (A) and nuclear extracts (B) were prepared, and PAGE was done as described in Materials and Methods. Blots were analyzed with antibodies to AR, calpain, calpastatin, and actin. The study was repeated twice, and representative data are shown.

Figure 6.

AR cleavage is associated with changes in calpain and calpastatin levels during the progression of synchronized LNCaP cells from G0-G1 to S phase. LNCaP cells were synchronized by isoleucine deprivation as described in Materials and Methods. Following release from isoleucine block, cells were harvested (trypsinized) either at various times corresponding to key phases of the cell cycle (0 hour, G0-G1; 4–8 hours, early G1; 12–16 hours, late G1; and 20–24 hours, S phase) or from exponentially growing cultures (Expo). Cytosol extracts (A) and nuclear extracts (B) were prepared, and PAGE was done as described in Materials and Methods. Blots were analyzed with antibodies to AR, calpain, calpastatin, and actin. The study was repeated twice, and representative data are shown.

Close modal

The levels of calpain and calpastatin also varied during the cell cycle. Cytosolic calpain levels were highest early in the cell cycle, decreased in late G1, and increased as S phase approached (Fig. 6A). Cytosolic calpastatin levels roughly mirrored calpain levels (Fig. 6A). The pattern of increasing cleavage of AR to ATD fragments seems to correlate to the increasing levels of calpain. Nuclear calpain was lowest during early G1 (Fig. 6B) and increased as the cell cycle progressed, correlating with the progressive increase in AR ATD fragments. Calpastatin levels, on the other hand, remained relatively steady throughout the cell cycle, although the level of the two highest molecular weight forms fluctuated.

In summary, there seems to be a correlation, more pronounced in the nucleus, between increases in calpain expression during progression through the cell cycle and the appearance of AR ATD fragments. This suggests that calpain might be responsible for AR cleavage as cells enter S phase. Although calpastatin is present in both nuclear and cytosolic extracts, greater variation is seen in cytosolic levels of calpastatin, suggesting different effects in the two compartments.

UV light activates caspase-3, triggers apoptosis in LNCaP cells, and causes decreased AR levels but does not generate ATD fragments. Caspase-3 has been implicated in the proteolytic cleavage of AR, but the experiments described above failed to reveal activated caspase-3 in exponentially growing or synchronized LNCaP cells. Therefore, we exposed LNCaP cells to doses of UV radiation known to trigger programmed cell death in cultured cancer cells and examined the effect on activation of caspase-3 and AR. Twelve hours after UVB exposure, LNCaP cells showed a mild degree of rounding up but little detachment from their substratum; by 24 hours after exposure, the apoptotic pathway was activated as evidenced by rounding and detachment of ∼20% of the cells at the higher UV dose (data not shown). There was significant, UV dose-dependent cleavage of PARP (Fig. 7A), a classic marker of programmed cell death. Although UV treatment did not affect the level of intact caspase-3 (Fig. 7B , top), it led to the appearance of cleaved caspase-3 fragments. This confirms that caspase-3 is present only in the inactive form in untreated, exponentially growing, or synchronized LNCaP cells but that it can be generated under specific conditions.

Figure 7.

Exponentially growing LNCaP cells were exposed to either 300 or 600 J/m2 of UV radiation; cells were scraped into medium 12 or 24 hours later, and whole-cell extracts were prepared and subjected to PAGE. A, Western blots were incubated with antibody to PARP to determine if the apoptotic process was initiated (top) and with the pAb N20 to the ATD of AR (middle) or antibody to actin to assess uniformity of loading (bottom). B, blots were incubated with antibody to intact caspase-3 (top) or antibody to cleaved caspase-3 (bottom). Results are from a single experiment.

Figure 7.

Exponentially growing LNCaP cells were exposed to either 300 or 600 J/m2 of UV radiation; cells were scraped into medium 12 or 24 hours later, and whole-cell extracts were prepared and subjected to PAGE. A, Western blots were incubated with antibody to PARP to determine if the apoptotic process was initiated (top) and with the pAb N20 to the ATD of AR (middle) or antibody to actin to assess uniformity of loading (bottom). B, blots were incubated with antibody to intact caspase-3 (top) or antibody to cleaved caspase-3 (bottom). Results are from a single experiment.

Close modal

Although there was no change in the actin level, the level of AR was dramatically reduced by 24 hours after UVB irradiation at a dose of 600 J/m2 (Fig. 7A). However, this decrease in AR was not accompanied by an increase in ATD fragments of AR. Rather, compared with unexposed controls or low-dose UV at 24 hours, the 31/34-kDa ATD fragments were less prominent in cells undergoing programmed cell death.

Our studies are the first to show that Ca2+ stimulates AR breakdown in vitro and in vivo and that CaM bound to AR seems to play a role in calpain-mediated cleavage of AR in prostate cancer cells. When AR binds to CaM in the presence of 2 mmol/L Ca2+, AR breakdown is accelerated despite the presence of PMSF (Fig. 1). AR, calpain, and its inhibitor calpastatin all bind to CaM in a Ca2+-dependent manner and are eluted with the calcium chelator EGTA (Fig. 4). Similarly, AR immunoprecipitates contain CaM, calpain, and calpastatin (Fig. 5). In vitro, Ca2+ activates AR breakdown in both nuclear and cytosolic extracts, and the inhibitor of thiol proteases, ALLN, prevents Ca2+-stimulated AR breakdown (Fig. 2). Based on AR breakdown fragment sizes and AR antibody epitope locations, we propose a model of AR cleavage by calpain (see Fig. 8A). We suggest that calpain cleaves AR at protease-sensitive sites between the LBD and DBD (yielding an ∼75-kDa ATD fragment), between the ATD and DBD (yielding an ∼50-kDa ATD fragment), and approximately in the middle of the ATD (yielding a 31/34-kDa NH2-terminal fragment; Fig. 8A).

Figure 8.

Model of AR cleavage by calpain. A, structure of AR showing the location of pAb N20 and mAb 441 epitopes (amino acids 1–20 and 301–317, respectively) inferred calpain cleavage sites (arrows) that yield 76-kDa, ∼50-kDa, and 31/34-kDa fragments, all three of which bind to CaM in a Ca2+-dependent manner. aa, amino acid; Q, polymorphic glutamine repeat (48); P, proline repeat; G, polymorphic glycine repeat (48); H, hinge region; AF, activation function; FQNLF motif (amino acids 23–27; ref. 49). B, model of interactions between AR, CaM, calpain, and calpastatin and the role of Ca2+ in calpain-mediated cleavage of AR. At low concentrations, calpain, calpastatin, and CaM bind to the AR presumably at CaM-binding sites on the AR; calpain is enzymatically inactive under these conditions. At elevated Ca2+ concentrations, calpain becomes activated (change in conformation), calpastatin dissociates [or is cleaved by activated calpain (37, 50)], and calpain cleaves AR. This model suggests that CaM-binding sites on the AR are at calpain cleavage sites.

Figure 8.

Model of AR cleavage by calpain. A, structure of AR showing the location of pAb N20 and mAb 441 epitopes (amino acids 1–20 and 301–317, respectively) inferred calpain cleavage sites (arrows) that yield 76-kDa, ∼50-kDa, and 31/34-kDa fragments, all three of which bind to CaM in a Ca2+-dependent manner. aa, amino acid; Q, polymorphic glutamine repeat (48); P, proline repeat; G, polymorphic glycine repeat (48); H, hinge region; AF, activation function; FQNLF motif (amino acids 23–27; ref. 49). B, model of interactions between AR, CaM, calpain, and calpastatin and the role of Ca2+ in calpain-mediated cleavage of AR. At low concentrations, calpain, calpastatin, and CaM bind to the AR presumably at CaM-binding sites on the AR; calpain is enzymatically inactive under these conditions. At elevated Ca2+ concentrations, calpain becomes activated (change in conformation), calpastatin dissociates [or is cleaved by activated calpain (37, 50)], and calpain cleaves AR. This model suggests that CaM-binding sites on the AR are at calpain cleavage sites.

Close modal

Proteins that bind to Ca2+ and/or are activated by Ca2+-binding proteins, such as CaM, act as decoders of the Ca2+ signal. In vitro, Ca2+ is known to change calpain conformation, effecting its autocatalytic cleavage and activation (34). Calpain heavy and light chains both have CaM homology domains (35, 36). Calpastatin binds to these CaM homology domains and inactivates calpain (37). Interestingly, virtually all proteins that bind to CaM are also substrates for calpain (38). Calpain, calpastatin, and CaM exist in a complex with AR (Fig. 5), and Ca2+ stimulates AR degradation (Fig. 3) in LNCaP cells. As depicted in Fig. 8B, these observations raise the intriguing possibility that CaM, bound to AR, recruits calpain and calpastatin to cleavage sites on AR. At low Ca2+, CaM-bound calpain is enzymatically inactive. However, at high Ca2+, the conformation of the calpain-CaM-calpastatin complex is altered in such a way that calpain is released from the inhibitory effect of calpastatin, thereby allowing calpain to cleave AR (Fig. 8B). This model is also consistent with our earlier observation that the anti-CaM drug N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, which can alter CaM conformation, induces AR degradation in LNCaP cells (28).

Ca2+, as a second messenger, enables cells to walk a tightrope between life and death. In exponentially growing rat prostate cancer cells, increasing intracellular Ca2+ shunts cells into G0-G1 phase and induces the growth arrest gene gadd153 (39). Thapsigargin (3941), β-lapachone (42), and ionomycin (40, 43) all induce striking increases in intracellular Ca2+ in prostate cancer cells and trigger programmed cell death. This process is accompanied by growth arrest (39) and down-regulation of AR expression (40). Our studies show that, both in cultured LNCaP cells and in cell-free extracts, increasing Ca2+ allowed AR degradation whereas decreasing Ca2+ (by chelation either in vitro with EDTA or intracellularly with AM-BAPTA) attenuated AR breakdown (Figs. 2 and 3). Thus, an increase in intracellular Ca2+ both suppresses the expression and stimulates the breakdown of proteins necessary for proliferation and viability of prostate cancer cells.

That AR is a substrate for calpain is not surprising. Estrogen receptor (44) and progesterone receptor (45) are cleaved by what we now know to be calpain. In LNCaP cells, cleavage of β-catenin, a key molecule in the Wnt signaling pathway that controls AR expression, is mediated by calpain (46). In this study, we observed that AR levels inversely correlated with calpain levels during the cell cycle in LNCaP cells; when AR levels were increasing, calpain levels were low in early G1 phase, whereas as AR levels were beginning to decline, calpain levels were at a maximum in S phase (Fig. 6).

Caspase-3 does not seem to be the enzyme that cleaves AR under physiologic conditions. AR with expanded polyglutamine repeats has been implicated in neurodegenerative disease wherein AR is cleaved between the DBD and LBD into a toxic, “truncated” ATD-DBD moiety (18). Caspase-3 is suggested to be the enzyme responsible for the cleavage of this pathologic AR (1922). Based on these observations of AR in neurodegenerative diseases, caspase-3 is implicated to cleave AR in prostate cancer cells (15, 23). However, we found that, under physiologic conditions, caspase-3 is not present in its activated form in exponentially growing LNCaP cells and caspase-3 is not part of the AR-CaM complex. Furthermore, activation of caspase-3 following irradiation of LNCaP cells with UVB radiation is not associated with cleavage of AR into 76-kDa, 50-kDa, or 31/34-kDa ATD fragments. Thus, caspase-3 could play a role in AR breakdown either directly or indirectly only under stress conditions; under normal growth conditions, it is an unlikely candidate for AR degradation during the cell cycle.

The degradation of AR is complex and multifaceted, and different proteolytic systems seem to be involved in transcription of genes regulated by AR, degradation of damaged AR, progress through the cell cycle, and programmed cell death. Although initial studies with proteasome inhibitors suggested that the proteasomal pathway was a major route for degradation of AR (5), subsequent studies (7, 9) have indicated that the primary effect of proteasome inhibitors in prostate cancer cells is to alter the Akt pathway, thereby altering transcription and translation of AR. On the other hand, the proteasome is critical for AR transactivating activity [i.e., AR binding to the ARE (6, 8)]. This study adds to the complexity by implicating a proteolytic enzyme, calpain, novel to AR degradation but well established for cleaving key regulatory molecules. Certainly, breaking down AR by a Ca2+-dependent, CaM-interacting, calpastatin-regulated mechanism is consistent with our concept of the importance of protein-protein interactions during the cell cycle. The localization of CaM-binding site(s) to the ATD of AR focuses our attention on a region of AR ripe for targeted therapy development (47). We envision a critical regulatory role for a CaM-AR-calpastatin-calpain complex in the proliferation of prostate cancer cells and as targets of opportunity in the development of agents for treating prostate cancer.

Note: R.P. Pelley, K. Chinnakannu, and S. Murthy contributed equally to this work.

Grant support: NIH grant RO1-DK57864 and Department of Defense grant W81XWH-05-1-0071.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1
Gronberg H. Prostate cancer epidemiology.
Lancet
2003
;
361
:
859
–64.
2
Zegarra-Moro OL, Schmidt LJ, Huang H, Tindall DJ. Disruption of androgen receptor function inhibits proliferation of androgen-refractory prostate cancer cells.
Cancer Res
2002
;
62
:
1008
–13.
3
Reddy GP, Barrack ER, Dou QP, et al. Regulatory processes affecting androgen receptor expression, stability, and function: potential targets to treat hormone-refractory prostate cancer.
J Cell Biochem
2006
;
98
:
1408
–23.
4
Voges D, Zwickl P, Baumeister W. The 26S proteasome: a molecular machine designed for controlled proteolysis.
Annu Rev Biochem
1999
;
68
:
1015
–68.
5
Sheflin L, Keegan B, Zhang W, Spaulding SW. Inhibiting proteasomes in human HepG2 and LNCaP cells increases endogenous androgen receptor levels.
Biochem Biophys Res Commun
2000
;
276
:
144
–50.
6
Kang Z, Pirskanen A, Janne OA, Palvimo JJ. Involvement of proteasome in the dynamic assembly of the androgen receptor transcription complex.
J Biol Chem
2002
;
277
:
48366
–71.
7
Gaughan L, Logan IR, Neal DE, Robson CN. Regulation of androgen receptor and histone deacetylase 1 by Mdm2-mediated ubiquitylation.
Nucleic Acids Res
2005
;
33
:
13
–26.
8
Lin HK, Altuwaijri S, Lin WJ, et al. Proteasome activity is required for androgen receptor transcriptional activity via regulation of androgen receptor nuclear translocation and interaction with coregulators in prostate cancer cells.
J Biol Chem
2002
;
277
:
36570
–6.
9
Lin HK, Wang L, Hu YC, Altuwaijri S, Chang C. Phosphorylation-dependent ubiquitylation and degradation of androgen receptor by Akt require Mdm2 E3 ligase.
EMBO J
2002
;
21
:
4037
–48.
10
Wilson EM, French FS. Effects of proteases and protease inhibitors on the 4.5 S and 8 S androgen receptor.
J Biol Chem
1979
;
254
:
6310
–9.
11
de Boer W, Bolt J, Kuiper GG, Brinkmann AO, Mulder E. Analysis of steroid- and DNA-binding domains of the calf uterine androgen receptor by limited proteolysis.
J Steroid Biochem
1987
;
28
:
9
–19.
12
Kallio PJ, Janne OA, Palvimo JJ. Agonists, but not antagonists, alter the conformation of the hormone-binding domain of androgen receptor.
Endocrinology
1994
;
134
:
998
–1001.
13
Kuiper GG, de Ruiter PE, Trapman J, et al. Localization and hormonal stimulation of phosphorylation sites in the LNCaP-cell androgen receptor.
Biochem J
1993
;
291
:
95
–101.
14
Abdullah A, Trifiro MA, Panet-Raymond V, et al. Spinobulbar muscular atrophy: polyglutamine-expanded androgen receptor is proteolytically resistant in vitro and processed abnormally in transfected cells.
Hum Mol Genet
1998
;
7
:
379
–84.
15
Gregory CW, He B, Wilson EM. The putative androgen receptor-A form results from in vitro proteolysis.
J Mol Endocrinol
2001
;
27
:
309
–19.
16
Duff J, McEwan IJ. Mutation of histidine 874 in the androgen receptor ligand-binding domain leads to promiscuous ligand activation and altered p160 coactivator interactions.
Mol Endocrinol
2005
;
19
:
2943
–54.
17
Kuil CW, Berrevoets CA, Mulder E. Ligand-induced conformational alterations of the androgen receptor analyzed by limited trypsinization. Studies on the mechanism of antiandrogen action.
J Biol Chem
1995
;
270
:
27569
–76.
18
Butler R, Leigh PN, McPhaul MJ, Gallo JM. Truncated forms of the androgen receptor are associated with polyglutamine expansion in X-linked spinal and bulbar muscular atrophy.
Hum Mol Genet
1998
;
7
:
121
–7.
19
Kobayashi Y, Miwa S, Merry DE, et al. Caspase-3 cleaves the expanded androgen receptor protein of spinal and bulbar muscular atrophy in a polyglutamine repeat length-dependent manner.
Biochem Biophys Res Commun
1998
;
252
:
145
–50.
20
Wellington CL, Ellerby LM, Hackam AS, et al. Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract.
J Biol Chem
1998
;
273
:
9158
–67.
21
Ellerby LM, Hackam AS, Propp SS, et al. Kennedy's disease: caspase cleavage of the androgen receptor is a crucial event in cytotoxicity.
J Neurochem
1999
;
72
:
185
–95.
22
LaFevre-Bernt MA, Ellerby LM. Kennedy's disease. Phosphorylation of the polyglutamine-expanded form of androgen receptor regulates its cleavage by caspase-3 and enhances cell death.
J Biol Chem
2003
;
278
:
34918
–24.
23
Lee DK, Chang C. Endocrine mechanisms of disease: expression and degradation of androgen receptor: mechanism and clinical implication.
J Clin Endocrinol Metab
2003
;
88
:
4043
–54.
24
Grad JM, Lyons LS, Robins DM, Burnstein KL. The androgen receptor (AR) amino-terminus imposes androgen-specific regulation of AR gene expression via an exonic enhancer.
Endocrinology
2001
;
142
:
1107
–16.
25
van Laar JH, Bolt-de Vries J, Zegers ND, Trapman J, Brinkmann AO. Androgen receptor heterogeneity and phosphorylation in human LNCaP cells.
Biochem Biophys Res Commun
1990
;
166
:
193
–200.
26
Kuiper GG, de Ruiter PE, Brinkmann AO. Androgen receptor heterogeneity in LNCaP cells is caused by a hormone independent phosphorylation step.
J Steroid Biochem Mol Biol
1992
;
41
:
697
–700.
27
Waller AS, Sharrard RM, Berthon P, Maitland NJ. Androgen receptor localisation and turnover in human prostate epithelium treated with the antiandrogen, casodex.
J Mol Endocrinol
2000
;
24
:
339
–51.
28
Cifuentes E, Mataraza JM, Yoshida BA, et al. Physical and functional interaction of androgen receptor with calmodulin in prostate cancer cells.
Proc Natl Acad Sci U S A
2004
;
101
:
464
–9.
29
Cifuentes E, Croxen R, Menon M, Barrack ER, Reddy GP. Synchronized prostate cancer cells for studying androgen regulated events in cell cycle progression from G1 into S phase.
J Cell Physiol
2003
;
195
:
337
–45.
30
Subramanyam C, Honn SC, Reed WC, Reddy GP. Nuclear localization of 68 kDa calmodulin-binding protein is associated with the onset of DNA replication.
J Cell Physiol
1990
;
144
:
423
–8.
31
Byeon SW, Pelley RP, Ullrich SE, et al. Aloe barbadensis extracts reduce the production of interleukin-10 after exposure to ultraviolet radiation.
J Invest Dermatol
1998
;
110
:
811
–7.
32
Nazareth LV, Stenoien DL, Bingman WE III, et al. A C619Y mutation in the human androgen receptor causes inactivation and mislocalization of the receptor with concomitant sequestration of SRC-1 (steroid receptor coactivator 1).
Mol Endocrinol
1999
;
13
:
2065
–75.
33
Santella L. The role of calcium in the cell cycle: facts and hypotheses.
Biochem Biophys Res Commun
1998
;
244
:
317
–24.
34
Strobl S, Fernandez-Catalan C, Braun M, et al. The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium.
Proc Natl Acad Sci U S A
2000
;
97
:
588
–92.
35
Emori Y, Kawasaki H, Sugihara H, et al. Isolation and sequence analyses of cDNA clones for the large subunits of two isozymes of rabbit calcium-dependent protease.
J Biol Chem
1986
;
261
:
9465
–71.
36
Emori Y, Kawasaki H, Imajoh S, Kawashima S, Suzuki K. Isolation and sequence analysis of cDNA clones for the small subunit of rabbit calcium-dependent protease.
J Biol Chem
1986
;
261
:
9472
–6.
37
Wendt A, Thompson VF, Goll DE. Interaction of calpastatin with calpain: a review.
Biol Chem
2004
;
385
:
465
–72.
38
Wang KK, Villalobo A, Roufogalis BD. Calmodulin-binding proteins as calpain substrates.
Biochem J
1989
;
262
:
693
–706.
39
Lin XS, Denmeade SR, Cisek L, Isaacs JT. Mechanism and role of growth arrest in programmed (apoptotic) death of prostatic cancer cells induced by thapsigargin.
Prostate
1997
;
33
:
201
–7.
40
Gong Y, Blok LJ, Perry JE, Lindzey JK, Tindall DJ. Calcium regulation of androgen receptor expression in the human prostate cancer cell line LNCaP.
Endocrinology
1995
;
136
:
2172
–8.
41
Skryma R, Mariot P, Bourhis XL, et al. Store depletion and store-operated Ca2+ current in human prostate cancer LNCaP cells: involvement in apoptosis.
J Physiol
2000
;
527
Pt 1:
71
–83.
42
Tagliarino C, Pink JJ, Reinicke KE, et al. μ-Calpain activation in β-lapachone-mediated apoptosis.
Cancer Biol Ther
2003
;
2
:
141
–52.
43
Zhu N, Wang Z. Calreticulin expression is associated with androgen regulation of the sensitivity to calcium ionophore-induced apoptosis in LNCaP prostate cancer cells.
Cancer Res
1999
;
59
:
1896
–902.
44
Puca GA, Nola E, Sica V, Bresciani F. Estrogen binding proteins of calf uterus. Molecular and functional characterization of the receptor transforming factor: a Ca2+-activated protease.
J Biol Chem
1977
;
252
:
1358
–66.
45
Vedeckis WV, Freeman MR, Schrader WT, O'Malley BW. Progesterone-binding components of chick oviduct: partial purification and characterization of a calcium-activated protease which hydrolyzes the progesterone receptor.
Biochemistry
1980
;
19
:
335
–43.
46
Rios-Doria J, Kuefer R, Ethier SP, Day ML. Cleavage of β-catenin by calpain in prostate and mammary tumor cells.
Cancer Res
2004
;
64
:
7237
–40.
47
Wang G, Sadar MD. Amino-terminus domain of the androgen receptor as a molecular target to prevent the hormonal progression of prostate cancer.
J Cell Biochem
2006
;
98
:
36
–53.
48
Hakimi JM, Schoenberg MP, Rondinelli RH, Piantadosi S, Barrack ER. Androgen receptor variants with short glutamine or glycine repeats may identify unique subpopulations of men with prostate cancer.
Clin Cancer Res
1997
;
3
:
1599
–608.
49
He B, Kemppainen JA, Wilson EM. FXXLF and WXXLF sequences mediate the NH2-terminal interaction with the ligand binding domain of the androgen receptor.
J Biol Chem
2000
;
275
:
22986
–94.
50
Carafoli E, Molinari M. Calpain: a protease in search of a function?
Biochem Biophys Res Commun
1998
;
247
:
193
–203.