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[Cancer Research 65, 7717-7723, September 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Accumulation of p53 and Reductions in XIAP Abundance Promote the Apoptosis of Prostate Cancer Cells

Subhra Mohapatra, Baoky Chu, Xiuhua Zhao and W.J. Pledger

Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute and the University of South Florida Medical Center, Tampa, Florida

Requests for reprints: Subhra Mohapatra, H. Lee Moffitt Cancer Center and Research Institute, 129202 Magnolia Drive, Tampa, FL 33612. Phone: 813-745-6484; Fax: 813-979-6700; E-mail: mohapts{at}moffitt.usf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Toward the goal of developing effective treatments for prostate cancers, we examined the effects of cyclin-dependent kinase inhibitors on the survival of prostate cancer cells. We show that roscovitine, R-roscovitine, and CGP74514A (collectively referred to as CKIs) induce the apoptosis of LNCaP and LNCaP-Rf cells, both of which express wild-type p53. Apoptosis required caspase-9 and caspase-3 activity, and cytochrome c accumulated in the cytosol of CKI-treated cells. Amounts of p53 increased substantially in CKI-treated cells, whereas amounts of the endogenous caspase inhibitor XIAP decreased. CKIs did not appreciably induce the apoptosis of LNCaP cells treated with pifithrin-{alpha}, which prevents p53 accumulation, or of prostate cancer cells that lack p53 function (PC3 and DU145). Ectopic expression of p53 in PC3 cells for 44 hours did not reduce XIAP abundance or induce apoptosis. However, p53-expressing PC3 cells readily apoptosed when exposed to CKIs or when depleted of XIAP by RNA interference. These findings show that CKIs induce the mitochondria-mediated apoptosis of prostate cancer cells by a dual mechanism: p53 accumulation and XIAP depletion. They suggest that these events in combination may prove useful in the treatment of advanced prostate cancers.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The purine analogues roscovitine, R-roscovitine (CYC002), and CGP74514A inhibit the activitiy of cyclin-dependent kinases (CDKs), most notably cdk2, cdk1, and cdk7 (1–3). cdk2 and cdk1 promote the entry of cells into S phase and mitosis, respectively, and cdk7 facilitates transcription. Importantly, all three inhibitors induce the apoptosis of tumor cells (2, 4–9). How these inhibitors trigger apoptosis is, however, unclear.

Apoptosis requires the activation of a family of cysteine aspartyl proteases termed the caspases (10). Adaptor proteins promote the autocleavage and activation of initiator caspases (e.g., caspase-8 and caspase-9), initiator caspases cleave and activate effector caspases (e.g., caspase-3), and effector caspases induce a multiplicity of events that ultimately result in cell death (e.g., plasma membrane blebbing and DNA fragmentation). There are two apoptosis signaling pathways: the death receptor pathway and the mitochondrial pathway. When active, death receptors interact with adaptor proteins such as FADD and TRADD and activate caspase-8. When damaged, mitochondria release cytochrome c, which associates with the adaptor protein Apaf-1 and activates caspase-9. Most drugs signal apoptosis through the mitochondrial pathway.

Proteins that modulate caspase activity and thus determine whether cells live or die include the inhibitor of apoptosis proteins (IAPs) and the Bcl-2 proteins. The IAP family includes cIAP-1, cIAP-2, XIAP, and survivin (11). Of these proteins, XIAP is the most potent. IAPs interact with and inhibit the activity of processed caspases; thus, they function as "brakes" that can impede the apoptotic process once it begins. IAPs inactivate both initiator and effector caspases; caspase-9 and caspase-3 are IAP targets, whereas caspase-8 is not (12).

The Bcl-2 proteins are critical determinants of mitochondria-dependent caspase activation (13). Some Bcl-2 proteins are apoptotic (e.g., Bax, Bak, and the BH3-only proteins), whereas others are antiapoptotic (e.g., Bcl-2 and Bcl-XL). Death stimuli activate Bax and Bak, which perforate the outer mitochondrial membrane in a manner dependent on the BH3-only proteins. Although incompletely resolved, Bcl-2 and Bcl-XL prevent the activation of Bax and Bak by sequestering the BH3-only proteins or by interacting with Bax and Bak.

p53 is a transcription factor that often couples apoptotic signals to changes in the abundance and/or activity of the Bcl-2 proteins (14). It accumulates in cells in response to many chemotoxic drugs, typically as a result of stabilization, and it promotes apoptosis by both transcription-dependent and -independent mechanisms. In the nucleus, p53 transactivates genes encoding apoptotic proteins such as Bax and the BH3-only proteins Noxa and Puma (15–17). When localized to mitochondria, p53 activates Bax and Bak by interacting with Bcl-2 family members (18). The mitochondrial actions of p53 are newly described and incompletely characterized. Given its role as a death signal, it is not surprising that p53 is frequently mutated in human tumors (19).

Our studies examined the effects of roscovitine, R-roscovitine, and CG74514A on the survival of LNCaP cells, which are androgen-dependent prostate cancer cells that express wild-type p53. We show that these inhibitors induce the apoptosis of LNCaP cells by a dual mechanism: they increase the abundance of p53 and reduce the abundance of XIAP. Importantly, we also show that ectopic expression of p53 coupled with depletion of endogenous XIAP results in the death of androgen-independent prostate cancer cells that lack p53 function. Androgen independence is characteristic of advanced prostate tumors, which are typically both metastatic and drug resistant. Thus, our findings offer a potential means of eradicating a prevelant and often fatal form of cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents. The human prostate cancer cell lines LNCaP, PC3, and DU145 were provided by Dr. Wenlong Bai (University of South Florida). LNCaP-Rf cells were provided by Dr. Donald Tindall (Mayo Clinic). LNCaP and DU-145 cells were cultured in RPMI 1640 containing 10% FCS. PC3 cells were cultured in in DMEM containing 10% FCS. LNCaP-Rf cells were cultured in RPMI 1640 containing 10% charcoal-stripped FCS. All experiments were done on growing cells in medium containing 10% FCS.

Stocks of roscovitine, R-roscovitine, and CGP74514A were prepared in DMSO. Roscovitine, LY294002, and pifithrin-{alpha} were purchased from Calbiochem (La Jolla, CA). R-roscovitine, z-DEVD-fmk, and z-LEHD-fmk were obtained from Alexis Biochemicals (San Diego, CA). CGP74514A was purchased from Sigma (St. Louis, MO). Antibodies were obtained from Cell Signaling (Beverly, MA; Bad, PARP, and p53), Santa Cruz Biotechnology (Santa Cruz, CA; Bax), BD Transduction Laboratories (Lexington, KY; XIAP and Bcl-XL), Alpha Diagnostic (San Antonio, TX; survivin), Sigma (ß-actin and Flag), Upstate (Lake Placid, NY; Bak), Neomarker (Union City, CA; p21Cip1), and Oncogene (Uniondale, NY; PUMA).

Keratin 18 cleavage. Immunocytochemistry using the M30 antibody to the keratin 18 cleavage product was done according to the instructions of the manufacturer (Roche, Nutley, NJ). Twenty hours after plating in chamber slides, cells were incubated with roscovitine, R-roscovitine, CGP74514A, or DMSO (vehicle control). After incubation, cells were fixed in ice-cold methanol at 20°C for 30 minutes and incubated with FITC-conjugated M30 antibody for 60 minutes at room temperature. Cells were mounted with Vectashield containing 4',6-diamidino-2-phenylindole (DAPI).

DNA fragmentation. DNA fragmentation was determined using a photometric enzyme-linked immunoassay (Cell Death Detection ELISA kit, Roche) as specified by the manufacturer. Cells in 96-well plates were incubated in triplicate with roscovitine, R-roscovitine, CGP74514A, or DMSO and lysed for 30 minutes at room temperature. Cell lysates were incubated with biotin-conjugated anti-histone antibody and peroxidase-conjugated anti-DNA antibody on streptavidin-coated microtiter plates for 2 hours at room temperature. After incubation with ATBS substrate for development of color, absorbance was read on a spectrophotometer at 405 nm against ABTS solution as the blank. For background determination, lysis buffer was used instead of cell lysate.

Preparation of S100 extracts. Cells were washed in ice-cold PBS, resuspended in an isotonic mitochondrial buffer [210 mmol/L mannitol, 70 mmol/L sucrose, 1 mmol/L EDTA, 10 mmol/L HEPES (pH 7.5), and a protease inhibitor cocktail (Sigma)], and homogenized in a Dounce homogenizer. The heavy membrane fraction was removed by two successive centrifugations, the first at 1,000 x g for 10 minutes and the second at 10,000 x g for 10 minutes. To obtain the S100 fraction, the supernatant was centrifuged at 100,000 x g for 90 minutes.

Western blotting. Cells were rinsed with PBS and lysed in a buffer containing 50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 10% glycerol, 0.5% NP40, 1 mmol/L DTT, 0.1 mmol/L phenylmethylsulfonyl fluoride, 2.5 µg/mL leupeptin, 0.5 mmol/L NaF, and 0.1 mmol/L Na3VO4. After a 30 minute incubation, insoluble material was removed by centrifugation. Cell extracts normalized for amount protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked in PBS containing 0.05% Tween and 5% instant milk and incubated with antibody in PBS containing 0.05% Tween for 2 hours at room temperature. Proteins recognized by the antibody were detected by enhanced chemiluminescence using a horseradish peroxidase–coupled secondary antibody as specified by the manufacturer (Pierce, Rockford, IL).

Preparation of adenoviruses encoding p53, XIAP, and XIAP small interfering RNA. For preparation of XIAP small interfering RNA (siRNA), a double-stranded oligonucleotide (5'-GGCAGGTTGTAGATATATCAGCTCGAGCTGATATATCTACAACCTGCCCTTTTTG-3') was subcloned into pBluescript-U6 (provided by Dr. Yang Shi, Harvard University). The functionality of the resultant plasmid was ascertained by transient transfection of 293 cells. pBluescript-U6 with and without insert was digested with XbaI to remove the U6 promoter (used for production of control virus) and the U6 promoter plus the XIAP siRNA sequence. Excised DNA was subcloned into the pAdTrack shuttle vector. Shuttle vectors were recombined with pAdEasy-1 according to the instructions of the manufacturer (Stratagene, La Jolla, CA). 293 cells were transiently transfected with pAdEasy-1 to produce control adenovirus and adenovirus encoding XIAP siRNA. Viruses were expanded, purified by CsCl banding, and titered.

Adenovirus encoding Flag-XIAP or Flag-p53 (Pro 72 variant) was prepared using the pShuttle-CMV shuttle vector (Stratagene) and pAdEasy-1. Control virus was obtained by recombination of pShuttle-CMV without insert and pAdEasy-1.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CKIs induce the apoptosis of LNCaP cells and LNCaP-Rf cells. Roscovitine, R-roscovitine, and CGP74514A are referred to collectively as CKIs (for CDK inhibitors). Consistent with their inhibitory effects on cdk2 and cdk1 activity, CKIs blocked cell cycle progression when added to exponentially growing LNCaP cells. Less than 5% of cells were in S phase 20 hours after exposure to CKIs compared with ~40% of control (vehicle-treated) cells (data not shown). CKI-treated cells accumulated in both G1 and G2-M.

As indicators of apoptosis, we monitored the effects of CKIs on DNA fragmentation and caspase activation in LNCaP cells. All experiments were done on proliferating cells in serum-supplemented medium. Amounts of cytosolic, histone-associated DNA fragments were quantified by a photometric enzyme-linked immunoassay. As shown in Fig. 1A, DNA fragmentation was minimal in control cells and was 6- to 8-fold greater in cells receiving 25 µmol/L roscovitine or 5 µmol/L CGP74514A for 20 hours.



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  		Figure 1. Apoptosis of LNCaP cells by CKIs. A, LNCaP cells received the indicated concentrations of roscovitine or CGP74514A for 20 hours. Amounts of cytosolic, histone-associated DNA fragments were determined by a cell death detection ELISA. Bars, SD. bgd, background (buffer only). B, LNCaP cells received DMSO (control), 25 µmol/L roscovitine, or 25 µmol/L R-roscovitine for 20 hours. For detection of cells containing cleaved keratin 18 (K18), cells were immunostained with fluorescein-conjugated M30 antibody. For detection of all cells, cells were stained with DAPI. C, LNCaP cells received DMSO (control, C), 25 µmol/L roscovitine (ROS), 5 µmol/L CGP74514A (CGP), or 15 µmol/L LY294002 (LY) for 20 hours. Amounts of cleaved PARP were determined by Western blotting of cell extracts. Amounts of survivin are unaffected by CKIs (see Fig. 4A) and are shown as a loading control.

 


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  		Figure 4. p53 accumulation and XIAP depletion in CKI-treated LNCaP and LNCaP-Rf cells. A, LNCaP cells received the indicated amounts of roscovitine for 20 hours. B, LNCaP-Rf cells received DMSO (control, C), 25 µmol/L roscovitine (ROS), or 5 µmol/L CGP74514A (CGP) for 20 hours. C, LNCaP cells received 25 µmol/L roscovitine for times indicated. A-C, protein abundance was determined by Western blotting of cell extracts. ß-Actin serves as the loading control.

 
Caspase activity was indirectly assayed by monitoring the cleavage of two endogenous caspase substrates, keratin 18 and poly(ADP-ribose)polymerase (PARP). The M30 antibody specifically recognizes cleaved keratin 18 (20), and >30% of cells receiving 25 µmol/L roscovitine or 25 µmol/L R-roscovitine for 20 hours were M30 positive (Fig. 1B). All control cells were M30 negative. Western blotting showed PARP cleavage in cells exposed to roscovitine or CGP74514A for 20 hours (Fig. 1C). PARP was not cleaved in control cells or in cells exposed to LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K) activity. Although PI3K often contributes to cell survival and is active constitutively in LNCaP cells (21, 22), the lack of effect of LY294002 on LNCaP cells in serum-containing medium is in agreement with previous studies (22). We also found that CKIs do not inhibit AKT activity in LNCaP cells (data not shown).

Collectively, the data in Fig. 1 show that CKIs induce the apoptosis of LNCaP cells. As further indication of DNA fragmentation in CKI-treated LNCaP cells, we also found the following: (a) DNA from control cells migrated as a single band on agarose gels, whereas DNA from CKI-treated cells migrated as a series of smaller-sized bands (i.e., was laddered, data not shown). (b) As determined by flow cytotometry, >20% of CKI-treated cells contained a <2N content of DNA compared with 1% of control cells (data not shown). In all experiments, apoptosis was maximal 20 hours after addition of CKIs to cells (data not shown).

Additional studies showed apoptosis of LNCaP-Rf cells by CKIs. These androgen-independent cells were established by others by long-term culture of LNCaP cells in the absence of androgens (23). They express wild-type p53, as do LNCaP cells. The data in Fig. 2A show PARP cleavage in LNCaP-Rf cells exposed to 25 µmol/L roscovitine or 5 µmol/L CGP74514A for 20 hours. The data in Fig. 2B show keratin 18 cleavage in roscovitine-treated LNCaP-Rf cells. Apoptosis of CKI-treated LNCaP-Rf cells indicates that CKIs induce apoptosis in a manner unrelated to androgen dependence.



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  		Figure 2. Apoptosis of LNCaP-Rf cells by CKIs. A, LNCaP-Rf cells received DMSO (control, C), 25 µmol/L roscovitine (ROS), or 5 µmol/L CGP74514A (CGP) for 20 hours. Amounts of cleaved PARP were determined by Western blotting of cell extracts. B, LNCaP-Rf cells received DMSO (control) or 25 µmol/L roscovitine for 20 hours. Apoptosis was monitored by keratin 18 (K18) cleavage, as visualized by immunofluorescence using the M30 antibody.

 
CKI-induced apoptosis of LNCaP cells requires caspase-9 and caspase-3 activity. To identify the caspases that become active in LNCaP cells exposed to CKIs, we cotreated cells with roscovitine and a caspase-9 inhibitor (z-LEHD-fmk) or a caspase-3 inhibitor (z-DEVD-fmk) for 20 hours. Apoptosis was monitored by the DNA fragmentation assay. As shown in Fig. 3A, roscovitine did not induce apoptosis when presented to cells in combination with either inhibitor. Thus, the roscovitine-induced apoptosis of LNCaP cells requires caspase-9 activity and caspase-3 activity. The near complete rescue of LNCaP cells by the caspase-9 inhibitor suggests that CKIs activate primarily (if not exclusively) the mitochondrial pathway of apoptosis.



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  		Figure 3. Caspase activation and accumulation of cytochrome c in CKI-treated LNCaP cells. A, LNCaP cells were pretreated with the caspase-9 inhibitor z-LEHD-fmk or the caspase-3 inhibitor z-DEVD-fmk at the indicated concentrations for 1 hour. Roscovitine (ROS) was added to pretreated and nonpretreated cultures to a final concentration of 25 µmol/L. Cells were harvested 20 hours after addition of roscovitine, and amounts of cytosolic, histone-associated DNA fragments were determined. Bars, SD. bgd, background (buffer only) C, control (no roscovitine). B, LNCaP cells received DMSO (control) or 25 µmol/L roscovitine (ROS) for 20 hours. Amounts of cytochrome c in S100 extracts were determined by Western blotting.

 
To provide further evidence of mitochondrial dysfunction, we compared amounts of cytochrome c in mitochondria-free cytosolic extracts prepared from LNCaP cells treated with or without roscovitine for 20 hours. As shown in Fig. 3B, roscovitine-treated cells contained much more cytosolic cytochrome c than did control cells.

CKIs increase the abundance of p53 and reduce the abundance of XIAP in LNCaP cells. Toward the goal of determining the mechanism of CKI-induced apoptosis, we monitored amounts of several apoptosis-regulatory proteins in CKI-treated cells by Western blotting. Exposure of LNCaP cells to roscovitine (5-25 µmol/L) for 20 hours had no effect on the abundance of Bcl-XL, Bax, Bak, Bad, c-IAP-1, c-IAP-2, or survivin (Fig. 4A). Amounts of Bcl-2 were also unaffected by roscovitine (data not shown). On the other hand, amounts of XIAP declined >90% in cells receiving 25 µmol/L roscovitine. Conversely and in agreement with studies of other cell types (4, 8, 9, 24, 25), p53 increased in abundance in roscovitine-treated LNCaP cells. Similar results were obtained in LNCaP-Rf cells exposed to roscovitine or CGP74514A (Fig. 4B; data not shown).

The kinetics of p53 accumulation, XIAP depletion, and PARP cleavage in roscovitine-treated LNCaP cells are shown in Fig. 4C. p53 abundance increased within 2 hours of addition of roscovitine to cells and was maximal at 4 hours. p53 accumulation preceded PARP cleavage, which began 6 to 8 hours after roscovitine addition and increased thereafter. Increases in amounts of cleaved PARP parelleled decreases in amounts of XIAP. These kinetics are consistent with a model of apoptosis in which accumulation of the apoptotic protein p53 and loss of the antiapoptotic protein XIAP signal the death of CKI-treated prostate cancer cells.

p53-responsive genes include those encoding PUMA and the cell cycle inhibitor p21Cip1 (16, 26). Consistent with transcriptional activation of p53, both proteins accumulated in roscovitine-treated LNCaP cells, albeit with delayed kinetics relative to p53 accumulation (Fig. 4C). Delayed increases in p21Cip1 abundance in roscovitine-treated MCF-7 cells have been observed previously (9).

Efficient apoptosis of prostate cancer cells requires p53 accumulation. Our data suggest that CKIs induce the apoptosis of LNCaP and LNCaP-Rf cells by increasing amounts of p53 and/or by decreasing amounts of XIAP. To assess the p53-dependence of CKI-induced apoptosis, two sets of experiments were done. First, LNCaP cells were treated with roscovitine in the presence and absence of pifithrin-{alpha}, a compound that inhibits the accumulation and/or the transcriptional activity of p53 (27–29). As shown in Fig. 5A, p53 abundance did not increase in LNCaP cells cotreated with roscovitine and pifithrin-{alpha}. Importantly, pifithrin-{alpha} substantially reduced (but did not eliminate) PARP cleavage in roscovitine-treated cells. Thus, efficient apoptosis of LNCaP cells by CKIs requires increases in p53 abundance.



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  		Figure 5. Requirement for p53 for efficient apoptosis of CKI-treated prostate cancer cells. A, LNCaP cells received no addition (NA) or 30 µmol/L pifithrin-{alpha} (PTF{alpha}) for 24 hours. Cells subsequently received DMSO (control, C) or 25 µmol/L roscovitine (ROS) for 16 hours. Amounts of p53 and cleaved PARP were determined by Western blotting of cell extracts. B, LNCaP and PC3 cells received the indicated concentrations of roscovitine for 20 hours. Amounts of cytosolic, histone-associated DNA fragments were determined by a cell death detection ELISA. Bars, SD. bgd, background (buffer only). C, LNCaP and PC3 cells received the indicated concentrations of CGP74514A for 20 hours. Amounts of cleaved PARP and XIAP were determined by Western blotting of cell extracts. D, LNCaP and DU145 cells received the indicated concentrations of roscovitine for 20 hours. Amounts of cleaved PARP and XIAP were determined by Western blotting of cell extracts.

 
Second, prostate cancer cells that do not express p53 (PC3) or express mutant p53 (DU145) were exposed to CKIs for 20 hours. Such treatment produced small increases in amounts of fragmented DNA (Fig. 5B) and cleaved PARP (Fig. 5C-D). Overall, PC3 and DU145 cells were much less susceptible to CKI-induced apoptosis than were LNCaP cells. For example, amounts of fragmented DNA increased 8-fold in LNCaP cells receiving 25 µmol/L roscovitine but only 3-fold in PC3 cells receiving 50 µmol/L roscovitine (Fig. 5B). Exposure of PC3 and DU145 cells to CKIs for time periods >20 hours did not further increase DNA fragmentation or PARP cleavage (data not shown).

Collectively, the data in Fig. 5 show the p53 dependence of CKI-elicited prostate cancer cell apoptosis. Of interest, the data in Fig. 5C and D show robust reductions in XIAP abundance in PC3, DU145, and LNCaP cells in response to CKIs. This finding indicates that CKIs down-regulate XIAP by a p53-independent mechanism and that reductions in XIAP abundance are insufficient for maximal apoptosis.

XIAP depletion accelerates p53-induced apoptosis of prostate cancer cells. Although required for CKI-induced apoptosis, p53 accumulation may be insufficient. To assess sufficiency, we infected PC3 cells with adenovirus alone (control) or adenovirus encoding wild-type Flag-tagged p53 (Ad-p53). Twenty-four hours after addition of virus, cells received vehicle or CGP74514A; cells were harvested 20 hours thereafter (44 hours after addition of virus). Cells infected with Ad-p53 expressed Flag-p53 but did not apoptose (as monitored by PARP cleavage) in the absence of CGP7451A (Fig. 6A). Although not apoptotic in these conditions, Ad-p53 sensitized PC3 cells to CKI-induced apoptosis; as shown in Fig. 6A, cells receiving both Ad-p53 and CGP74514A contained more cleaved PARP than did cells receiving CGP74514A alone. These data suggest that p53 accumulation induces apoptosis in conjunction with additional CKI-elicited events. The data in Fig. 6A also show that ectopic expression of p53 does not reduce XIAP abundance, and results similar to those in Fig. 6A were obtained in experiments on DU145 cells (data not shown).



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  		Figure 6. Requirement for p53 accumulation and XIAP depletion for apoptosis of prostate cancer cells. A, PC3 cells were infected with adenovirus without insert (Ad) or Ad-p53 (500 virus particles per cell) for 24 hours. Cells then received DMSO (control, C) or 5 µmol/L CGP7514A (CGP) for 20 hours. Cell extracts were Western blotted with antibodies to Flag, PARP, and XIAP. B, PC3 cells were infected with Ad or Ad-p53 (500 virus particles per cell) for 24, 48, or 72 hours. Cell extracts were Western blotted with antibodies to PARP and p53. C, LNCap and PC3 cells were infected with Ad or Ad-p53 and coinfected with Ad-siXIAP (+) or control adenovirus (–) for 40 hours. Cell extracts were Western blotted with antibodies to Flag, PARP, and XIAP. LNCaP cells received adenovirus at 500 virus particles per cell. PC3 cells received adenovirus at 200 virus particles per cell. D, LNCaP cells were infected with Ad or adenovirus encoding Flag-tagged XIAP (Ad-XIAP; 500 virus particles per cell) for 40 hours. Cells then received DMSO (control, C) or 25 µmol/L roscovitine (ROS) for 12 hours. Cell extracts were Western blotted with antibodies to Flag and PARP.

 
Additional experiments showed PARP cleavage in PC3 cells exposed to Ad-p53 for 72 hours (Fig. 6B). In agreement with the data in Fig. 6A, Ad-p53 did not increase amounts of cleaved PARP (relative to control virus) when presented to cells for 24 or 48 hours. p53 was expressed in Ad-p53-infected cells at all time points. Thus, given enough time, ectopic p53 is apoptotic per se; however, it is a more efficient inducer of apoptosis when presented to cells in combination with CKIs.

CKIs may expedite p53-mediated apoptosis by reducing the abundance of XIAP. To test this hypothesis, we infected LNCaP and PC3 cells with Ad-p53, adenovirus encoding XIAP siRNA (Ad-siXIAP), or both. Cells expressing Ad-p53 expressed ectopic Flag-tagged p53, and expression of Ad-siXIAP substantially reduced XIAP abundance (Fig. 6C). Infection of cells with Ad-p53 or Ad-siXIAP for 40 hours did not induce apoptosis; however, cells readily apoptosed when coinfected with both viruses. These results suggest that apoptosis of prostate cancer cells by CKIs results from a combination of p53 accumulation and XIAP depletion.

As shown in Fig. 6D, overexpression of XIAP reduced the extent to which LNCaP cells apoptosed when exposed to roscovitine. This finding confirms the capacity of XIAP to promote the survival of CKI-treated prostate cancer cells. Overexpression of XIAP has been shown to inhibit the apoptosis of LNCaP cells by taxol and cisplatin (30, 31), as well as the apoptosis of other cancer cell types by various death stimuli (32–35).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies show that CKIs induce the apoptosis of prostate cancer cells that express wild-type p53 (LNCaP and LNCaP-Rf). CKIs activate the mitochondrial pathway of apoptosis in these cells, as indicated by (a) the abrogation of CKI-induced apoptosis by an inhibitor of caspase-9 and (b) the presence of cytochrome c in the cytosol of CKI-treated cells. Amounts of p53 increased substantially in cells exposed to CKIs, whereas amounts of XIAP decreased. We suggest that these two events account in large part for the apoptotic effects of CKIs on prostate cancer cells.

Maximal apoptosis of prostate cancer cells by CKIs required the accumulation of wild-type p53. First, pifithrin-{alpha} prevented p53 accumulation and inhibited apoptosis when added to LNCaP cells in combination with roscovitine. Others observed similar effects of pifithrin-{alpha} in 5-azacytidine-treated colon cancer cells and doxirubucin-treated endothelial cells (28, 29). Second, p53-deficient prostate cancer cells (PC3 and DU145) apoptosed to a much lesser extent when exposed to CKIs than did LNCaP cells. PC3 and DU145 cells were not, however, completely resistant to CKI-induced apoptosis nor were pifithrin-{alpha}-treated LNCaP cells. Thus, CKIs also induce apoptosis, albeit ineffectively, by a p53-independent mechanism. Whether this mechanism involves other members of the p53 family remains to be determined.

Prostate cancer cells ectopically expressing p53 or depleted of XIAP by RNA interference did not apoptose, as monitored 40 to 44 hours after infection. Thus, within this time frame, neither event is sufficient for apoptosis. On the other hand, cells efficiently apoptosed within 44 hours when coinfected with both Ad-p53 and Ad-XIAP siRNA. These findings show that p53 accumulation and XIAP depletion, both of which occur in CKI-treated cells, recapitulate the apoptotic actions of CKIs. We note that these events are independent; XIAP depletion does not alter p53 abundance, ectopic expression of p53 does not alter XIAP abundance, and CKIs reduce XIAP abundance in the absence of functional p53.

PC3 cells apoptosed when exposed to Ad-p53 alone for 72 hours. Thus, in agreement with several previous studies on prostate cancer cells (36–40), p53 can induce apoptosis by itself. However, as shown here, XIAP depletion clearly accelerates the onset of p53-induced apoptosis. Insufficiency of p53 accumulation and of XIAP loss for apoptosis is supported by other studies. Colletier et al. (41) showed that Ad-p53 sensitized LNCaP cells to radiation-induced death but was not apoptotic per se. Amantana et al. (42) found that XIAP depletion was weakly apoptotic for DU145 cells but synergized with cisplatin (41). Carson et al. (43) showed caspase activation in LNCaP cells microinjected with both cytochrome c and Smac but not with either alone (36). Smac binds and inactivates IAPs (11), and XIAP is the predominant Smac binding partner in LNCaP cells (43).

CKIs inhibit the activity of cdk2, cdk1, and cdk7 (1–3). Which of these kinases (if any) modulates the abundance of p53 and XIAP, and how it does so, remains to be determined. cdk7 promotes transcriptional elongation by phosphorylating RNA polymerase II (44). Thus, ablation of cdk7 activity by CKIs may impede production of the transcripts for XIAP and for Mdm2, the protein that signals p53 destruction (45). Consistent with this hypothesis, we and others observed reduced amounts of XIAP mRNA and Mdm2 mRNA in roscovitine-treated MT2 and MCF-7 cells, respectively (8, 9). cdk2 and cdk1 phosphorylate p53 at Ser315 (46–48), and studies by Sakaguchi et al. (49) suggest that p53 degrades more rapidly when phosphorylated at this site. On the other hand, replacement of Ser315 with alanine did not negate p53 accumulation in roscovitine-treated MCF-7 cells (9). Although cdk7 also phosphorylates p53 (50, 51), its effects on p53 half-life are not known.

Prostate cancer is the most common malignancy in males in Europe and North America (52). In the United States, >200,000 men develop prostate cancer each year and >30,000 men die from prostate cancer each year. In initial stages, prostate cancers are localized and androgen-dependent; in advanced stages, they are invasive, metastatic, and heterogeneous in terms of androgen dependence. The standard treatment for advanced prostate cancer is androgen ablation. Such treatment induces the apoptosis of androgen-dependent cells and reduces the size of primary and metastatic lesions; however, the surviving androgen-resistant cells typically form aggressive drug-resistant tumors. These observations emphasize the need for effective treatments for advanced prostate cancer, and we suggest that expression of wild-type p53 coupled with depletion of XIAP may aid in this endeavor.


   Acknowledgments
 
Grant support: Cortner-Couch Endowed Chair for Cancer Research (W.J. Pledger), NIH grant CA93544 (W.J. Pledger), Florida Department of Health James and Esther King Biomedical Research Grant, American Cancer Society Institutional Research Grant (H. Lee Moffitt Cancer Center and Research Institute), and American Cancer Society Florida grant (S. Mohapatra).

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.

We thank Nancy Olashaw for article preparation and the Cytometry, Molecular Biology, and Molecular Imaging Core Laboratories at the Moffitt Cancer Center for helpful service.

Received 2/ 1/05. Revised 5/13/05. Accepted 6/ 9/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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