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[Cancer Research 64, 1377-1385, February 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Induction of Androgen Receptor-Dependent Apoptosis in Prostate Cancer Cells by the Retinoblastoma Protein

Xintao Wang, Haiyun Deng, Indranil Basu and Liang Zhu

Department of Developmental and Molecular Biology, The Albert Einstein Comprehensive Cancer Center, Albert Einstein College of Medicine, Bronx, New York


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Re-expression of a tumor suppressor in tumor cells that lack it is an effective way to study its functional activities. However, because tumor cells contain multiple mutations, tumor suppressor functions that are dependent on (an)other regulators are unlikely to be identified by its re-expression alone if the other regulators are also mutated. In this study, we show that re-expression of retinoblastoma (RB) together with the androgen receptor (AR) in RB- and AR-deficient prostate cancer DU-145 cells resulted in an apoptotic activity, acting through the mitochondria damage-initiated caspase activation pathway, which was not present when RB, or the AR, was re-expressed alone. The ability of RB + AR to induce mitochondria damage was dependent on the proapoptotic proteins Bax and Bak and could be blocked by the antiapoptotic protein Bcl-xL. Coexpressed AR did not detectably change RB’s regulation of E2F and cell cycle progression in culture. On the other hand, coexpressed RB could activate the transactivation activity of the AR in an androgen-depleted media. Although androgen induced greater AR transactivation activity in this condition, it did not induce apoptosis in the absence of coexpressed RB. Analysis of mutants of RB and the AR indicated that intact pocket function of RB and the transactivation activity of the AR were required for RB + AR-induced apoptosis. These results provide direct functional data for an AR-dependent apoptosis-inducing activity of RB and highlight the importance of cell type-specific regulators in obtaining a more complete understanding of RB.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The tumor suppressor retinoblastoma (RB) and the RB-E2F pathway play central roles in cell proliferation and tumorigenesis (see Ref. 1 for a recent review). RB functions as a transducer between the cell cycle engine and the cellular gene expression programs, most significantly the E2F-regulated gene expression program, to control cell proliferation at various cell cycle transition points, most importantly at the G1-S transition. In addition to this general, non-cell type-specific role, it is generally believed that RB also has cell type-specific functions. This latter aspect of RB, although much less well understood, is clearly an integral part of RB function because RB mutations in cancer have cell type-specific patterns. RB mutations are primarily found in retinoblastoma, osteosarcoma, lung, prostate, breast, and bladder cancers (2) .

A commonly used and very effective approach to revealing the functional activities of tumor suppressors is to re-express them in tumor cells that lack them because of mutations selected for during the tumorigenesis process. Re-expression of RB in RB-deficient tumor cells generally leads to repression of E2F and G1 cell cycle arrest; and data from the re-expression experiments constitute a major part in the current model of RB function as mentioned above. A hallmark of tumorigenesis, however, is that cancer cells almost always contain multiple alterations (3) . Based on this fact, re-expression of a tumor suppressor in the absence of other presumably important regulators (because they are also mutated in tumorigenesis) is unlikely to reveal all of the functional activities of this particular tumor suppressor (because certain functions of this tumor suppressor may depend on certain other regulators).

We have applied these considerations to the study of RB in prostate cancer cells. Combined results from many studies have demonstrated that the RB gene is mutated in about 20% of the prostate cancer samples at both early and late stages of this disease (4, 5, 6, 7) . The widely used prostate cancer cell model DU145 contains exon 21 deletion in its RB gene (4) , which abolishes the pocket functions of RB (therefore considered as a null mutation). Re-expression of wild-type RB in DU145 cells did not have cell cycle effects in culture (4) . The androgen receptor (AR) plays important roles in prostate cell proliferation, differentiation, and survival and is often mutated in prostate cancer, particularly in late stages (8, 9, 10) . DU145 cells do not express the AR. It has been demonstrated previously that RB and the AR can physically interact and RB can stimulate AR-mediated transactivation (11 , 12) . In this study, we determined the effects of re-expressing RB and the AR separately and in combination in DU145 cells. This experimental approach revealed an AR-dependent apoptosis-inducing activity of RB.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
Prostate cancer cell lines DU145 and LNCaP were purchased from American Type Culture Collection and cultured in DMEM media containing 10% fetal bovine serum (Invitrogen), 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Charcoal dextran treated fetal bovine serum was purchased from Gemini, dihydrotestosterone (DHT) from Sigma, and the synthetic androgen R1881 from DuPont Merck Pharmaceutical Co. Androgen antagonist Casodex (bicalutamide) was a gift from AstraZeneca (Cheshire, United Kingdom). DU145 cells were transfected with pUHD-172neo (encoding the reverse tetracycline-regulated transactivator rtTA; provided by Dr. H. Bujard). G418-resistant clonal cell lines were established and screened for their ability to express a reporter gene under the control of the tetracycline-responsive promoter (pUHD10–3; provided by Dr. H. Bujard). These clones are named DU-rtTA. DU-rtTA cells were then stably transfected with pUHD10–3-RB (13) and pBabePuro to establish puromycin-resistant DU-RB cell lines. Induction of RB expression in DU-RB cell lines was achieved by treating these cells with doxycycline hydrochloride (Dox, Sigma) at 1 µg/ml. DU-RB cells were subsequently transfected with pCMV-ARzeo or pCMV-AR-KAzeo [the AR and AR-KA (mutation of K630 to A) cDNAs were provided by Dr. R. Pestell] to establish zeocin (0.1 µg/ml final concentration) resistant DU-RB-AR and DU-RB-KA cell lines, respectively. For analysis of RB mutant {Delta}22, identical methods were used to generate DU145 cell lines that inducibly express RB{Delta}22 and constitutively express the AR (DU-{Delta}22-AR).

Cell Proliferation and Apoptosis Assays.
BrdUrd pulse labeling was performed with a final concentration of 50 nM for 4 h with cells cultured on glass cover slips in various conditions. Cells were fixed with cold ethanol, denatured with 2 N HCl, and stained with anti-BrdUrd (Calbiochem) and secondary goat antimouse IgG conjugated with FITC. BrdUrd-positive cells were identified under fluorescent microscope and the percentage of BrdUrd-positive cells in the whole population determined. Photograph of cultured cells under a phase-contrast microscope and the determination of cell numbers were conducted with standard procedures. To measure apoptosis with sub-G1 fluorescence-activated cell sorter analysis, induced cells (both floating and attached) were harvested, fixed in ethanol, stained with propidium iodide, and analyzed by a fluorescence-activated cell sorter Scan with CellQuest software. Green fluorescent protein-Annexin V staining was performed with a kit from Clonetech. To measure mitochondrial membrane potential, cells were incubated for 30 min in 40 nM DiOC6 (3,3'-dihexyloxacarbocyanine iodide; Molecular Probes) before harvest. Total cells were then harvested, washed with PBS, and analyzed by fluorescence-activated cell sorter. Bcl-xL-expressing adenovirus was provided by Dr. J. D. Molkentin. Virus stocks were prepared by the CsCl purification method, and the amounts of viruses needed to infect >80% of the cells were determined by immunofluorescence staining of Bcl-xL. Cells were first infected with adenovirus for 24 h and then induced to express RB as usual. When apoptosis was induced by chemicals, cells were treated with okadaic acid at 20 nM or etopside at 50 µM for 24 h. To determine the functional status of cellular p53, cells were treated with actinomycin D at 20 nM for 24 h.

Immunoprecipitation and Western Blot Assays.
Western blot analysis was conducted as described previously (14) except for the detection of caspases and poly(ADP-ribose) polymerase, for which we followed the protocol described previously (15) . For immunoprecipitation experiments, cell extracts prepared from two 10-cm plates of cells (containing 1000 µg of total protein) were incubated with 2 µg of antibody on ice for 4 h, and immuno-complexes were precipitated with 25 µl of a mix of protein A and G beads in 4°C with rocking. The immunoprecipitates were washed three time with the lysis buffer (250 mM NaCl, 50 mM HEPES (pH 7.0), 5 mM EDTA, 0.1% NP40, 2 mM phenyl methylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM DTT, 10 µg/ml Leupeptin, 10 µg/ml Aprotinin, 20 µg/ml trypsin inhibitor, 1 mM Benzamidine) before being analyzed by Western blotting. Antibodies against the AR (C-19 and 441), Bax (N-20), Bak (G-23), Bcl-2 (N-19), Bcl-xL (M-125), cyclin E (C-19), cyclin A (H432), Cdk2 (M-2), Cdk4 (C-22), Cdc2 (17) , Cdc25A (F-6), p27 (C19), p21 (F-5), and p53 (DO-1) were purchased from Santa Cruz Biotechnology; poly(ADP-ribose) polymerase (9542), caspase 3 (9661), caspase 9 (9502), and caspase 8 (9746) were obtained from Cell Signaling; and RB (clone G3–245) was purchased from BD PharMingen. Anti-E2F1 (KH-20) was provided by Dr. K. Helin.

Promoter Reporter Assays.
DU145 cells were plated in 6-well plates at 5 x 105 per well in fetal bovine serum (FBS) media. One day after plating, the cells were transfected with LipofectAMINE Plus with 3 µg of total DNA. The E2F reporter E2F-TK-Luc and the AR reporter mouse mammary tumor virus-Luc were provided by Dr. R. Pestell. The expression vector for a full-length RB with all potential phosphorylable Thr/Ser changed to Ala was provided by Dr. J. W. Harper. At the end of a 6 h transfection period, cells were switched to charcoal dextran-treated serum (CDT) media. When needed, DHT at 1 nM final concentration and Dox at 1 µg/ml final concentration were added for the last 7 h. R1881 was also used to stimulate the AR, which yielded similar results as DHT (data not shown). Casodex at 20 µM was used as androgen antagonist. Cell lysates were prepared by directly adding lysis buffer [25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N’,N',N',N'-tetraacetic acid, 10% glycerol, and 0.2% Triton X-100] to the cells on ice. Luciferase activity was determined with luciferase assay systems from Promega Corp. following the manufacturer’s protocol with an Autolumat LB 953 (EG&G Berthold). Luciferase activity was normalized for transfection efficiency with a cotransfected ß-galactosidase reporter.

Small Interfering (si)RNA-Mediated Knockdown of Bax and Bak.
DU-RB-AR cells were plated in 6-well plates and transfected with Oligofectamine (Invitrogen) according to the manufacturer’s protocol. siRNA against Bax and Bak were purchased from Dharmacon’s SMARTpool selection; these Bax and Bak siRNA pools are tested and proven to knockdown at lease by 75% of Bax and Bak, respectively. A negative control pool was included in all siRNA experiments. Forty-eight h after transfection, cells were split into fresh media with and without Dox for another 36 h before analysis.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Re-expression of RB and the AR and Their Physical Interaction in Prostate Cancer DU145 Cells.
We used prostate cancer cell model DU145, which lacks functional RB and the AR to study the functional relationships between these two regulators. Although previous experiments have established DU145-derived cell lines with constitutive re-expression of RB (suggesting that RB did not block cell cycle progression in DU145 cells; Ref. 4 ), we still used inducible expression to avoid possible selection for cells that contain mutations that abolish the negative effects of RB during the generation of derivative cell lines. We transfected DU145 cells with pUHD-172neo to establish clonal lines that could express a test gene under the control of the tet-responsive promoter in the vector pUHD10–3 in response to Dox. Five such clones (DU-rtTA) were found to have this ability (data not shown).

DU145 cells are microsatellite mutator phenotype positive (16) . Defects in DNA mismatch repair in microsatellite mutator phenotype positive cells render them prone to replication errors. The human Bax gene contains a sequence of eight consecutive deoxyguanosines (the G-8 track) near its NH2 terminus, which is prone to mutations caused by DNA slippage during DNA replication. It was reported that the G-8 track of the Bax gene is homozygously mutated to G-9, and consequently the Bax protein is not expressed in DU145 cells (16) . In theory, the instability at the 8-G track should generate both wild type-to-mutant and mutant-to-wild type changes if no selective pressure is present to select against Bax-expressing cells. We determined Bax expression in the five DU-rtTA clones. As shown in Fig. 1ACitation , two of the five clones have restored Bax expression. We did not detect any difference in the proliferation rates among all these clones compared with the parental cells (data not shown). We picked clone 1 (the DU-rtTA cells) to establish subsequent cell lines and continued to monitor Bax expression. Bax expression was retained in all of the subsequent clones in this study (data not shown).



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  		Fig. 1. Restoration of Bax, retinoblastoma (RB), and androgen receptor (AR) expression in DU145 prostate cancer cells and physical interaction between RB and the AR. A, total cell extracts from parental DU145 and five DU-rtTA clonal lines were Western blotted with an anti-Bax antibody. B, equal amounts of total cell extracts from LNCaP, DU-RB, and DU-RB-AR cell lines were Western blotted with antibodies against RB and the AR. RB expression was induced by the addition of doxorubicin (Dox) in the culture media for 16 h. C, total extracts of DU-RB and DU-RB-AR cells, uninduced or induced by Dox as indicated, were either analyzed directly by Western blotting (the "total" lanes) or immunoprecipitated first with RB or AR antibodies [the "RB IP" and "AR IP" lanes]. In the left panel, the cells were incubated in media containing fetal bovine serum, whereas in the right panel, cells were incubated in media containing charcoal dextran-treated serum (CDT) with or without the added synthetic androgen R1881 at 1 nM for 48 h. IP, immunoprecipitation.

 
We then transfected the DU-rtTA cells with pUHD10–3-RB (13) to establish clones that could be induced to express RB. A total of four independent cell lines were established and were found to exhibit similar properties. Data from a representative one, DU-RB (Fig. 1B)Citation , were chosen for presentation in this report. From the DU-RB cells, we further derived lines that contained constitutive expression of the AR by stably transfecting DU-RB cells with pcDNA3-ARzeo to create a pair of DU145 cells (DU-RB-AR cells; Fig. 1BCitation ) that only re-express the AR (DU-RB-AR cells without induction of RB) and that re-express both the AR and RB (DU-RB-AR cells with induction of RB). Again, one representative clone of three was presented in this report. When equal amounts of total proteins were compared, the AR and Dox-induced RB levels in DU-RB-AR cells were about 3-fold lower and 2-fold higher, respectively, than those in the LNCaP cells, which express functional RB and the AR and are androgen responsive and androgen dependent for proliferation (Fig. 1B)Citation .

Previous studies have demonstrated that RB and the AR could physically interact in vitro with purified RB and the AR proteins, and in vivo in mammalian two-hybrid assays (11 , 12) . RB interacted with the NH2-terminal part of the AR, and this interaction was independent on the AR ligand. With our inducible cell lines, we could now demonstrate in vivo interaction of RB and the AR through coimmunoprecipitation (Fig. 1C)Citation . This interaction was also independent on the AR ligand. These results suggest a direct relationship between RB and the AR in DU145 cells.

Combined Re-expression of RB and the AR Induces Apoptosis.
Inducible re-expression of RB (DU-RB cells with Dox) did not result in inhibition of cell proliferation compared with uninduced cells in parallel, as measured by cell number determination, BrdUrd labeling, and cell morphology (Fig. 2, A–C)Citation . DU145 cells with constitutive re-expression of the AR (DU-RB-AR cells without induction of RB) showed a slight reduction in proliferation activity compared with the parental cells as measured by cell number determination and BrdUrd labeling (Fig. 2, A and B)Citation . We further determined whether re-expression of the AR, in the absence of RB induction, rendered DU145 cells androgen responsive by comparing their proliferation after 2 days of culture in media containing charcoal dextran-treated FBS (CDT media) and CDT media containing 1 nM DHT. BrdUrd labeling at the end of the 2 day period showed same labeling indices for media containing FBS, CDT, or CDT + DHT (data not shown). These results demonstrate that re-expression of RB or the AR alone did not have significant effects on DU145 cell proliferation in the conditions used.



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  		Fig. 2. Apoptosis caused by re-expression of retinoblastoma (RB) and the androgen receptor (AR) in DU145 cells. DU-rtTA, DU-RB, and DU-RB-AR cells were treated with doxorubicin (Dox), or untreated in parallel, as indicated, for 24 h. Total numbers of attached cells were counted (A) after the plates were photographed under a phase-contrast microscope (C). Cells were pulse-labeled with BrdUrd for 4 h, fixed, stained with anti-BrdUrd, and the number of BrdUrd-positive cells determined (B). Total cells (including the floating cells) were fixed in ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry (D). Percentages of cells in the sub-G1 region were indicated. Total cells were stained with green fluorescent protein-Annexin V and PI without ethanol fixation and analyzed by flow cytometry (E). Cells in the bottom right window were defined as apoptotic because they were Annexin V-stained positive but PI-stained negative (PI did not leak through the cell membrane of live cells). Cells in the top left window were defined as death through necrosis. Cells appeared in the top right window in the late stages of apoptosis because of secondary cell membrane damage.

 
We then studied the effects of re-expressing RB together with the AR by inducing DU-RB-AR cells in FBS media. Twenty-four h after RB induction, we observed a significant reduction in the numbers of attached cells compared with uninduced cultures in parallel (Fig. 2, A and C)Citation . This reduction in cell numbers was apparently not caused by a block to enter S phase as measured by BrdUrd incorporation (Fig. 2B)Citation but by cell death through apoptosis. As shown in Fig. 2DCitation , RB induction in DU-RB-AR cells resulted in the appearance of sub-G1 cell fractions in flow cytometry analysis and the externalization of phosphatidylserine on the plasma membrane as measured by Annexin V staining (Fig. 2E)Citation . Clearly, the combined re-expression of RB and the AR created an apoptotic activity, which was not present when RB or the AR was re-expressed alone.

RB + AR Affects Mitochondria Integrity to Induce Apoptosis.
Apoptoic mechanisms can be generally divided into mitochondria-dependent and -independent groups. To determine the involvement of mitochondria in RB + AR-induced apoptosis, we measured the mitochondria membrane integrity by DiOC6 staining. DiOC6 is selectively retained in the mitochondria when the mitochondria membrane potential is intact. Fig. 3ACitation shows that re-expression of RB led to the loss of mitochondria retention of DiOC6. The extent of this loss was greater than those induced by chemicals that are widely used to induce mitochondria-mediated cell death such as etopside and okadaic acid. As shown later in Fig. 6BCitation , induction of RB in DU-RB cells with Dox did not cause mitochondria damage.



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  		Fig. 3. Involvement of the mitochondria-initiated caspase activation pathway in retinoblastoma (RB) + androgen receptor (AR)-induced apoptosis. A, DU-RB-AR cells were induced with doxorubicin (Dox), or treated with the indicated apoptosis-inducing chemicals without Dox, for 24 h. Total cells were then incubated with DiOC6 for 30 min and analyzed by flow cytometry. Cells in the lower-brightness peak contained damaged mitochondria. B, DU-RB and DU-RB-AR cells were treated with Dox, or okadaic acid in the absence of Dox, as indicated, for 24 h. Cell extracts were made from total cells and analyzed by Western blotting with the indicated antibodies. Molecular weights of full-length and cleaved caspases are marked. C, DU-RB and DU-RB-AR cells were induced with Dox as indicated, and protein levels of Bcl-2, Bax, and Bcl-xL in total extracts were determined with Western blotting with the respective antibodies. Expression of Bcl-xL after infection of DU-RB-AR cells with a Bcl-xL-expressing adenovirus was also shown. D, DU-RB-AR cells were first infected with adenoviruses expressing either LacZ or Bcl-xL for 24 h and then induced with Dox for another 24 h. Western blotting was performed as in B. E, DU-RB-AR cells were treated as in D, harvested, and analyzed for sub-G1 fraction as in Fig. 2DCitation . PARP, poly(ADP-ribose) polymerase; OA, okadaic acid.

 


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  		Fig. 6. Biochemical effects of retinoblastoma (RB) on the androgen receptor (AR). A, DU-RB and DU-RB-AR cells were transfected with an AR reporter plasmid [mouse mammary tumor virus (MMTV)-AR] and were incubated in charcoal dextran-treated serum (CDT) media containing dihydrotestosterone (DHT; 1 nM) or vehicle (ethanol) and with or without doxorubicin (Dox). Luciferase activity was measured and presented as relative units. Triplicate samples were analyzed for every data point and SDs displayed as error bars. The experiments were repeated three times with similar results. B, DU-RB and DU-RB-AR cells were incubated in different media as indicated and induced with Dox for 24 h. Cells were then stained with DiOC6 and analyzed as in Fig. 3ACitation . Experiments in C and D were performed in identical ways as in A and B, respectively, but including a parallel group with the treatment of Casodex (20 µM) as indicated. EtOH, ethanol; FBS, fetal bovine serum.

 
We next determined the activation status of several key caspases by their cleavage and the cleavage of the caspase substrate poly(ADP-ribose) polymerase. As shown in Fig. 3BCitation , RB induction caused significant cleavage of poly(ADP-ribose) polymerase, caspase 3, 9, and 8. The extent of these effects was similar to those induced by okadaic acid. The activation of caspases 9 and 3 are consistent with the well-established mitochondria-initiated caspase activation pathway (mitochondria damage -> cytochrome C release -> caspase 9 cleavage -> caspase 3 cleavage). The cleavage of caspase 8 could be mediated by other activated caspases (including caspases 9 and 3). Alternatively, caspase 8 cleavage could be caused by the activation of the death receptor pathway or other mechanisms upstream of mitochondria. The Bcl-2 family members Bcl-2, Bcl-xL, Bax, and Bak are the major regulators of mitochondria integrity (17) . Bcl-2 and Bcl-xL protect mitochondria membrane integrity whereas Bax and Bak induce mitochondria membrane depolarization. Bcl-2, Bax, and Bak protein levels did not change detectably after RB induction in both DU-RB and DU-RB-AR cells; but Bcl-xL proteins were not detectable (Fig. 3C)Citation . We therefore ectopically expressed Bcl-xL through an adenovirus vector in DU-RB-AR cells to determine the role of mitochondria damage in the cleavage of caspase 8 and apoptosis after RB induction. As shown in Fig. 3DCitation , adenovirus-mediated expression of Bcl-xL completely blocked cleavage of caspase 8 as well as caspases 9 and 3. Bcl-xL also completely prevented apoptosis (Fig. 3E)Citation . In the same experiments, the control LacZ adenovirus only slightly reduced caspase activation and sub-G1 fraction. We conclude from these results that RB + AR caused mitochondria damage to induce apoptosis.

RB + AR-Induced Apoptosis Requires Functions of the Proapoptosis Proteins Bax and Bak but Is Independent on p53.
Well-established mediators of mitochondria damage include the proapoptosis members of the Bcl-2 family Bax and Bak. These two proteins can self-oligomerize in mitochondria outer membrane to permeabilize it (18) . In the mouse, knockout of both Bax and Bak conferred resistance to normal apoptosis in animal development and in experimental apoptosis induced by many apoptosis-inducing agents, whereas knockout of Bax or Bak alone did not (19 , 20) . In human colon cancer cells, knockout of Bax alone resulted in complete resistance to apoptosis induced by non-steroidal anti-inflammatory drugs but no change in 5-fluorouracil and ceramide-induced apoptosis (21) . Lack of Bax expression was also shown to favor clonal selection in tumor growth when engrafted in nude mice (22) . The status and role of Bak were not addressed in these studies. The parental DU145 cells do not express Bax because of a frame-shift mutation, which was corrected during the establishment of our inducible cell lines (Fig. 1A)Citation .

We used siRNA-mediated knockdown to determine the roles of Bax and Bak in RB + AR-induced apoptosis, as shown in Fig. 4, A and BCitation . Knockdown of Bax or Bak (by >75%) did not affect the ability of RB + AR to induce apoptosis, suggesting that RB + AR-induced apoptosis is not dependent on Bax or Bak alone. When both Bax and Bak were subjected to knockdown, RB + AR-induced apoptosis was almost completely prevented (Fig. 4B)Citation . Thus, the apoptosis pathway activated by RB + AR involves Bax and Bak functions, which is consistent with our finding that ectopic expression of Bcl-xL was able to prevent RB + AR-induced apoptosis (Fig. 3)Citation because Bcl-xL antagonizes the activity of Bax and Bak.



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  		Fig. 4. Roles of the proapoptotic proteins Bax and Bak, and p53 in retinoblastoma (RB) + androgen receptor (AR)-induced apoptosis. A, DU-RB-AR cells were transfected with the indicated siRNA, induced with doxorubicin (Dox), and then analyzed by Western blotting to determine the protein levels of Bax, Bak, and actin. In B, DU-RB-AR cells treated in parallel were analyzed by DiOC6 labeling as in Fig. 3ACitation . C, U2OS and DU-RB-AR cells were treated with actinomycin D (20 nM) or Dox as indicated for 24 h. Total cell extracts were then analyzed by Western blotting to determine the levels of p53, p21, and actin. siRNA, small interfering RNA

 
We next determined whether RB + AR-induced apoptosis was dependent on p53. p53 of DU145 cells contains two point mutations Pro274-to-Leu and Val223-to-Phe (23) . To determine the functional status of this mutant p53, we treated DU-RB-AR cells with actinomycin D. Cells containing functional p53 (U2OS) were studied in parallel. As shown in Fig. 4CCitation , actinomycin D treatment induced p53 protein levels in both DU-RB-AR and U2OS cells, indicating that the signaling pathway to p53 and the ability of the mutant p53 in DU-RB-AR cells to stabilize are intact. However, the mutant p53 in DU-RB-AR cells was completely defective in its ability to transactivate the well-established p53 target gene p21Cip1 in response to actinomycin D treatment (Fig. 4C)Citation . These results suggest that RB + AR is able to induce apoptosis in the absence of a functional p53.

Biochemical Effects of RB and the AR on Each Other.
RB and the AR both have well established biochemical activities in regulation of gene expression. We investigated how RB and the AR affected the activities of each other in the prostate cancer DU145 cells. The best established function of RB is the repression of E2F. We determined the effects of RB induction on an E2F reporter in the absence (DU-RB cells) and presence (DU-RB-AR cells) of the AR; in the latter case, we also determined the effects of the AR ligand DHT. As shown in Fig. 5ACitation , induction of RB repressed the E2F reporter activity about 30% in DU-RB cells and about 50% in DU-RB-AR cells. The repression was not influenced by the presence or absence of the AR ligand DHT in DU-RB-AR cells in CDT media. When RB was expressed from transient transfection, which produced higher levels of RB proteins, repression of the E2F reporter was to a greater extent at about 60% in DU-RB cells and 50–60% in DU-RB-AR cells in the presence or absence of DHT. When an unphosphorylable RB was expressed by transient transfection, it led to further greater repression of E2F in both cell lines. This pattern of E2F repression by wild type and unphosphorylable RB in DU145-derived cell lines is similar to what has been observed after ectopic expression of RB in many other cell lines that are not sensitive to the G1-S arresting functions of RB.



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  		Fig. 5. Biochemical effects of the androgen receptor (AR) on retinoblastoma (RB). A, DU-RB and DU-RB-AR cells were transfected with an E2F-reporter plasmid (E2F-TK-Luc) together with the indicated expression vectors. RB{Delta}P is an unphosphorylable RB with phosphorylation sites mutated. After transfection, the cells were incubated in charcoal dextran-treated serum media with dihydrotestosterone (DHT; 1 nM) or vehicle (ethanol), and with or without doxorubicin (Dox) as indicated. Luciferase activity was measured and presented as relative units. Triplicate samples were analyzed for every data point and SDs displayed as error bars. The experiments were repeated three times with similar results. B, total cell extracts of DU-RB and DU-RB-AR cells, with or without induction for 18 h, were analyzed with Western blotting with the indicated antibodies. Vec, empty vector; Cyc, cyclin; Cdc2, cyclin-dependent kinase 1; Cdk, cyclin-dependent kinase.

 
We also determined the effects of RB-mediated E2F repression on cellular genes. As shown in Fig. 5BCitation , proteins levels of several well-established RB-E2F repression target genes such as cyclin E, cyclin A, Cdk2, Cdc2, Cdc25A, and E2F1 itself were not detectably affected by the induction of RB in the absence or presence of the AR. Protein levels of Cdk4 and the cyclin-dependent kinase inhibitors p27 and p21 were also not affected by RB and the AR in these cells. The failure of induced RB to repress cellular E2F target genes could be caused by a number of factors (including deficiencies in histone deacetylases and/or chromatin remodeling proteins), and could be the reason for the inability of RB to cause G1-S block in these cells [(4) and Fig. 2BCitation ]. Nonetheless, data in Fig. 5Citation suggest that coexpression of the AR did not affect RB’s regulation of E2F in DU145 cells.

We measured the AR transactivation activity with the natural AR-responsive mouse mammary tumor virus-Luc reporter. As shown in Fig. 6ACitation , the AR in DU-RB-AR cells responded to DHT to similar extents as previous reports with transiently transfected AR in DU145 cells and in our DU-RB cell line (data not shown). Importantly, coexpression of RB in CDT media without added DHT significantly stimulated the transactivation activity of AR in the same assay. This stimulation of mouse mammary tumor virus-Luc was dependent on the AR because RB induction in DU-RB cells did not result in this stimulation. Dox also had no stimulatory effect on AR transactivation in the absence of RB (data not shown). Casodex, an androgen antagonist, inhibited DHT-induced AR transactivation activity by about 50%, which is consistent with reports in the literature (24 , 25) but did not reduce RB-induced AR transactivation activity in CDT media without added DHT (Fig. 6C)Citation , providing further support that RB can induce AR transactivation activity in the absence of androgen. When RB was induced in the presence of DHT, it did not affect DHT-stimulated AR transactivation activity.

The above results from inducible cell lines differed from pervious results obtained with transient transfection of the AR and RB, in which RB only stimulated AR transactivation activity in the presence of DHT (11 , 12) . Indeed, when we used transient transfection to express the AR and RB, we also found that RB only stimulated the AR in the presence of DHT. This difference between results from stable inducible cell lines and transient transfection is most likely caused by the different expression levels in these different experimental systems.

To determine whether the stimulation of AR transactivation activity by RB in the absence of added DHT was functionally relevant to the RB + AR-induced apoptosis, we measured mitochondria integrity after induction of RB in this condition. Fig. 6BCitation shows that RB induction in CDT media caused apoptosis to the same extent as in the FBS media. Addition of DHT only slightly increased apoptosis further. Fig. 6DCitation shows that addition of Casodex did not reduce RB + AR-induced mitochondria damage. Therefore, the stimulation of AR transactivation activity by RB in CDT media was sufficient for the induction of apoptosis. It is important to note here that DHT induced greater AR transactivation activity than RB but did not induce apoptosis in the absence of re-expressed RB.

AR Transactivation Activity and RB Pocket Functions Are Required for RB to Induce Apoptosis.
We used a genetic approach to determine whether the activation of the transactivation activity of the AR was required for RB to induce apoptosis. It was recently demonstrated that a K630-to-A mutation at the acetylation consensus site abrogated the transactivation activity of the AR (26) . We transfected the same DU-RB cell line with a pCDNA3-AR-KAzeo construct and selected with zeocin for clones with AR-KA expression, as we did for the DU-RB-AR cell line. A representative cell line (called DU-RB-KA for brevity) was shown in Fig. 7ACitation . Expression levels of the AR-KA protein in this cell line were the same as the levels of the wild-type AR expressed in the DU-RB-AR cell line, so were the levels of RB expression induced by Dox. As expected, the AR-KA in this cell line was unable to transactivate the mouse mammary tumor virus-Luc reporter in response to DHT (Fig. 7B)Citation . It also failed to be stimulated by RB induction. Induction of RB in this cell line did not induce mitochondria damage (Fig. 7C)Citation and DNA fragmentation (data not shown). We conclude that the transactivation activity of the AR is required for RB to induce apoptosis.



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  		Fig. 7. Requirement for the transactivation activity of the androgen receptor (AR) and the pocket functions of retinoblastoma (RB) in RB + AR-induced apoptosis. A, Western blotting of total cell extracts of DU-RB, DU-RB-AR, DU-RB-KA (expressing AR-K630A mutant), and DU-{Delta}22-AR (expressing RB{Delta}22 mutant) cells with anti-RB and anti-AR antibodies as in Fig. 1BCitation . B, DU-RB-AR, DU-RB-KA, and DU-{Delta}22-AR cells were transfected with the AR reporter plasmid mouse mammary tumor virus (MMTV)-AR and Luciferase activity analyzed as in Fig. 6ACitation . C, DU-RB-AR, DU-RB-KA, and DU-{Delta}22-AR cells cultured in fetal bovine serum media were induced with doxorubicin (Dox) for 24 h and analyzed with DiOC6 staining as in Fig. 3ACitation . EtOH, ethanol.

 
Most of the known activities of the RB protein are mediated through its pocket domain, and most of the naturally occurring mutations of RB map inside the pocket domain and disrupt its function. We established parallel cell lines expressing RB{Delta}22, the pocket functions of which are abolished by deletion of exon 22, to determine the relationship of RB + AR-induced apoptosis with the pocket domain. In this cell line (DU-{Delta}22-AR), protein levels of RB{Delta}22 after induction and the AR were similar to those in the DU-RB-AR cell line (Fig. 7A)Citation . As shown in Fig. 7BCitation , induction of RB{Delta}22 did not activate AR transactivation activity and (Fig. 7C)Citation did not induce apoptosis, demonstrating that the RB + AR-induced apoptosis is dependent on the pocket functions of RB. This result is consistent with previous findings that binding of viral onco-proteins (E1a, Tag, and E7) to the pocket domain blocked RB-AR interaction (12) . This result suggests that the ability of RB to activate the AR to induce apoptosis may be involved in the roles of RB as a tumor suppressor in prostate cancer cells.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although counter intuitive to the role of RB as a tumor suppressor, RB has been believed to have an antiapoptotic activity in a number of tissues based on phenotypes of the RB knockout mice (27, 28, 29) . Importantly however, recent studies with more advanced mouse genetic techniques have demonstrated that the apoptosis observed in RB knockout mouse embryos could largely be attributed to non-cell-autonomous effects of inactivating RB in the whole animal. Perhaps most striking is the demonstration that most of the defects observed previously in RB knockout embryos could be prevented by supplying the RB knockout embryo with normal placenta (30) . It was shown that RB inactivation led to increased proliferation, not apoptosis, of trophoblasts resulting in defective nutrient transport from the mother to the embryo. These recent findings clearly indicate the need to re-evaluate previous interpretations of the apoptosis phenotypes in various tissues of RB knockout embryos.

In this study we identified an apoptotic activity of RB by re-expressing RB together with the AR in a prostate cancer cell model that is deficient for both RB and the AR. This functional identification significantly improves our knowledge of RB in prostate cells obtained from a large amount of previous studies of this subject.

Roles of RB and the AR in Prostate Cell Apoptosis in Vivo.
A well-known physiological apoptotic response in prostate epithelial cells takes place after castration (31) , indicating that androgen-AR signaling is antiapoptotic whereas the non- or low-androgen-stimulated AR may be apoptotic. It was shown that immediately before the onset of apoptosis, RB expression increased significantly in prostate epithelial cells, implicating a proapoptosis role of RB in prostate epithelial cells (32) . Reconstitution of prostate tissue with RB knockout prostate epithelial cells in an otherwise RB wild-type host revealed that RB inactivation led to hyperplasia of prostate epithelium, not apoptosis (33) . When these animals were subjected to pharmacological doses of androgen and estrogen, RB-deficient prostate tissues are more prone to oncogenic transformation without increases in apoptosis, providing genetic evidence for an antiproliferative, but not antiapoptotic, role of RB in prostate epithelial cells (33) . On the other hand, transgenic expression of the AR in prostate epithelial cells through a probasin promoter in an otherwise wild-type mouse led to both proliferation and apoptosis resulting in no net hyperplasia, demonstrating that the AR signaling can promote both proliferation and apoptosis (34) . Precancerous nodules were observed only in focal regions indicating that additional mutations, most likely those that abolish apoptosis, are necessary for oncogenic transformation. RB mutation could be one such additional mutation, which would be consistent with the presence of RB mutations in a significant portion of prostate cancers in humans. Our discovery that the combined action of RB and the AR can lead to apoptosis may provide a mechanistic explanation for these in vivo observations and a cell model to study the apoptotic roles of RB and the AR in prostate cells.

Roles of RB and the AR in Prostate Cell Apoptosis in Vitro.
The roles of RB and the AR in apoptosis have been separately studied in cultured prostate cancer cells. The AR has been shown to play a proapoptosis role in prostate cancer cells in a number of scenarios. When re-expressed in the prostate cancer cell line PC-3 (which contains RB), androgen could induce apoptosis (35) . Brca1 could further stimulate the androgen-activated AR transactivation activity to enhance apoptosis in these cells (36) . The AR has also been shown to play an apoptotic role in prostate cancer cells in the absence of coexpressed RB. Overexpression of activated MEKK1 (mitogen-activated protein kinase kinase kinase 1) required the AR to cause apoptosis in DU145 cells (25) . Like Brca1, MEKK1 stimulated AR transactivation activity and induced apoptosis in an androgen-dependent manner. These studies have led to the current belief that "super-activation" of the AR can cause apoptosis. A unique feature of RB + AR-induced apoptosis demonstrated in our study is that RB could activate AR transactivation activity and induce apoptosis in media containing charcoal dextran-treated (CDT) serum without added androgen. More importantly, we showed that androgen-stimulated AR transactivation activity to a greater degree than RB but did not induce apoptosis in the absence of coexpressed RB. Thus, it is unlikely that RB-induced apoptosis by super-activating AR transactivation activity. This characteristic of RB + AR-induced apoptosis is more consistent with the currently known in vivo prostate cell apoptosis as discussed above. Pharmacological doses of androgen caused hyperplasia, but not apoptosis, in the reconstituted RB-/- prostate tissues (33) ; prostate cell apoptosis occurred without the need for high dose of androgen in probasin-AR transgenic mice (34) , and prostate epithelial cells underwent apoptosis after castration (androgen withdrawal; Ref. 31 ).

Overexpression of RB in cultured cells generally leads to inhibition of cell cycle progression but caused apoptosis in prostate cancer LNCaP cells (32) , which contain RB and the AR. It was also proposed that RB might be required for apoptosis of prostate cancer cells induced by cell detachment and protein kinase C activation because these treatments led to caspase activation and apoptosis in LNCaP cells but not in DU145 cells (37) . Interestingly however, it is known that fibroblasts respond to these same treatments with G1 cell cycle arrest, not apoptosis. The reason for these differences has not been understood. Based on our finding, we suggest that the presence of the AR signaling pathway in prostate cells could be responsible for these differences.

In another study, it was shown that constitutive re-expression of RB alone in DU145 cells sensitized these cells to killing by {gamma}-irradiation (38 , 39) . Interestingly, this cell killing in the absence of the coexpressed AR did not involve caspase activation. Rather, a serine protease was involved. We have demonstrated that RB + AR-induced apoptosis in DU145 cells is through mitochondria-initiated caspase pathway, a cell death pathway activated in most, if not all, apoptotic responses to many signals including DNA damage, oncogenic stimuli, and disruptions to metabolism and intracellular trafficking. Our findings that RB stimulated AR transactivation and apoptosis in the absence of added androgen; androgen stimulated AR transactivation activity to a greater extent but did not induce apoptosis in the absence of RB; and RB did not induce apoptosis when co-re-expressed with a transactivation-defective AR mutant suggest that RB may stimulate the AR to activate expression of a set of genes to induce apoptosis, and this set of genes are different from those activated by the AR and androgen. Currently, almost all studies of cellular AR target genes have been done with the prostate cancer cell line LNCaP (40 , 41) , because this cell line shows androgen responsiveness for proliferation and expression of the well-established AR target gene prostate-specific antigen (42) . The kinase inhibitor p21Cip1 has been recently demonstrated as a direct AR target gene in LNCaP cells (43) , but conflicting results were obtained in another study with the same LNCaP cells (44) . It is clear that many key AR target genes remain to be identified. A major difficulty in identifying AR target genes is that although ectopically expressed AR is functional as measured by Androgen receptor Response Element (ARE)-containing promoter reporter plasmids, it generally does not lead to androgen responsiveness of the currently known cellular AR target genes. This is indeed the case with AR re-expression in DU145 cells. The AR target gene prostate-specific antigen responded robustly to androgen in LNCaP cells but was not detectably stimulated by androgen in the DU-RB-AR cells under the same conditions (our unpublished results). This phenomenon indicates that a lot remains to be learned about the cofactors that are involved in AR-mediated transcription regulation of cellular genes. RB may serve as one cofactor to stimulate AR transactivation activity for a specific set of genes that regulate the mitochondria-mediated apoptotic pathway.

Underlying Mechanisms for the Opposing Effects of the AR in Apoptosis.
Increased apoptosis of prostate epithelial cells after castration perhaps represents the most physiologically relevant evidence that the AR can play opposing roles in the regulation of prostate cell survival, because this phenomenon suggests that androgen-AR signaling is antiapoptotic whereas the non- or low-androgen-stimulated AR is apoptotic. How, mechanistically, could the same AR play opposing roles in prostate cell survival? Re-expression of the tumor suppressor PTEN (phosphatase and tensin homologue deleted from chromosome 10) in LNCaP cells causes apoptosis, which is countered by androgen-AR signaling (45) . In this system, it was demonstrated that the androgen-activated AR represses the forkhead transcription factor FKHR (the target genes of which include proapoptotic proteins such as the Fas ligand and Bim) to inhibit apoptosis. Castration also induces increased apoptosis in vertebral osteoblasts (46) . With the osteoblasts (and extended to mouse embryo fibroblasts and human HeLa cells), Kousteni et al. (46) reported that both androgen and estrogen receptors have sex-nonspecific ligand-dependent antiapoptotic activity, which depends on a cytoplasmic function of the receptors to stimulate Src/Shc/Erk signaling. Based on these studies, it appears that the antiapoptotic activity of androgen-AR signaling may not depend on the transactivation activity of the AR. In this study, we showed that the RB + AR-induced apoptosis is dependent on the transactivation activity of the AR (as measured on a adenine and uridine-rich element promoter reporter). These findings together suggest that the opposing effects of the AR in prostate cell survival, at least in the experimental scenarios used, are based on distinct molecular activities of the AR.


   ACKNOWLEDGMENTS
 
We thank Drs. Mark Czaja, Hermann Bujard, J. Wade Harper, Kristian Helin, Rick Kitsis, Jeffery Molkentin, and Richard Pestell for reagents and advice.


   FOOTNOTES
 
Grant support: United States Army Prostate Cancer Research Program (DAMD17-00-1-0074).

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.

Notes: X. Wang and H. Deng contributed equally to this work. L. Zhu is a scholar of the Leukemia and Lymphoma Society of America.

Requests for reprints: Liang Zhu, Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Room U-519, Bronx, New York 10461. Phone: (718) 430-3320; Fax: (718) 430-8975; E-mail: lizhu{at}aecom.yu.edu

Received 8/ 5/03. Revised 11/21/03. Accepted 12/17/03.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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