Research Article 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 Abstract activates caspase-9. Most drugs signal apoptosis through the Toward the goal of developing effective treatments for mitochondrial pathway. prostate cancers, we examined the effects of cyclin-dependent Proteins that modulate caspase activity and thus determine kinase inhibitors on the survival of prostate cancer cells. We whether cells live or die include the inhibitor of apoptosis proteins show that roscovitine, R-roscovitine, and CGP74514A (collec- (IAPs) and the Bcl-2 proteins. The IAP family includes cIAP-1, cIAP- tively referred to as CKIs) induce the apoptosis of LNCaP and 2, XIAP, and survivin (11). Of these proteins, XIAP is the most potent. LNCaP-Rf cells, both of which express wild-type p53. Apoptosis IAPs interact with and inhibit the activity of processed caspases; required caspase-9 and caspase-3 activity, and cytochrome c thus, they function as ‘‘brakes’’ that can impede the apoptotic accumulated in the cytosol of CKI-treated cells. Amounts of process once it begins. IAPs inactivate both initiator and effector caspases; caspase-9 and caspase-3 are IAP targets, whereas caspase- p53 increased substantially in CKI-treated cells, whereas amounts of the endogenous caspase inhibitor XIAP decreased. 8 is not (12). CKIs did not appreciably induce the apoptosis of LNCaP cells The Bcl-2 proteins are critical determinants of mitochondria- treated with pifithrin-A, which prevents p53 accumulation, or dependent caspase activation (13). Some Bcl-2 proteins are of prostate cancer cells that lack p53 function (PC3 and apoptotic (e.g., Bax, Bak, and the BH3-only proteins), whereas DU145). Ectopic expression of p53 in PC3 cells for 44 hours did others are antiapoptotic (e.g., Bcl-2 and Bcl-XL). Death stimuli not reduce XIAP abundance or induce apoptosis. However, activate Bax and Bak, which perforate the outer mitochondrial p53-expressing PC3 cells readily apoptosed when exposed to membrane in a manner dependent on the BH3-only proteins. CKIs or when depleted of XIAP by RNA interference. These Although incompletely resolved, Bcl-2 and Bcl-XL prevent the findings show that CKIs induce the mitochondria-mediated activation of Bax and Bak by sequestering the BH3-only proteins or by interacting with Bax and Bak. apoptosis of prostate cancer cells by a dual mechanism: p53 accumulation and XIAP depletion. They suggest that these p53 is a transcription factor that often couples apoptotic signals events in combination may prove useful in the treatment of to changes in the abundance and/or activity of the Bcl-2 proteins advanced prostate cancers. (Cancer Res 2005; 65(17): 7717-23) (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 Introduction the nucleus, p53 transactivates genes encoding apoptotic proteins The purine analogues roscovitine, R-roscovitine (CYC002), and such as Bax and the BH3-only proteins Noxa and Puma (15–17). CGP74514A inhibit the activitiy of cyclin-dependent kinases When localized to mitochondria, p53 activates Bax and Bak by (CDKs), most notably cdk2, cdk1, and cdk7 (1–3). cdk2 and cdk1 interacting with Bcl-2 family members (18). The mitochondrial promote the entry of cells into S phase and mitosis, respectively, actions of p53 are newly described and incompletely characterized. and cdk7 facilitates transcription. Importantly, all three inhibitors Given its role as a death signal, it is not surprising that p53 is induce the apoptosis of tumor cells (2, 4–9). How these inhibitors frequently mutated in human tumors (19). trigger apoptosis is, however, unclear. Our studies examined the effects of roscovitine, R-roscovitine, Apoptosis requires the activation of a family of cysteine and CG74514A on the survival of LNCaP cells, which are androgen- aspartyl proteases termed the caspases (10). Adaptor proteins dependent prostate cancer cells that express wild-type p53. We promote the autocleavage and activation of initiator caspases show that these inhibitors induce the apoptosis of LNCaP cells by a (e.g., caspase-8 and caspase-9), initiator caspases cleave and dual mechanism: they increase the abundance of p53 and reduce activate effector caspases (e.g., caspase-3), and effector caspases the abundance of XIAP. Importantly, we also show that ectopic induce a multiplicity of events that ultimately result in cell death expression of p53 coupled with depletion of endogenous XIAP (e.g., plasma membrane blebbing and DNA fragmentation). There results in the death of androgen-independent prostate cancer cells are two apoptosis signaling pathways: the death receptor pathway that lack p53 function. Androgen independence is characteristic of and the mitochondrial pathway. When active, death receptors advanced prostate tumors, which are typically both metastatic and interact with adaptor proteins such as FADD and TRADD and drug resistant. Thus, our findings offer a potential means of activate caspase-8. When damaged, mitochondria release cyto- eradicating a prevelant and often fatal form of cancer. chrome c, which associates with the adaptor protein Apaf-1 and Materials and Methods Cell culture and reagents. The human prostate cancer cell lines LNCaP, Requests for reprints: Subhra Mohapatra, H. Lee Moffitt Cancer Center and PC3, and DU145 were provided by Dr. Wenlong Bai (University of South Research Institute, 129202 Magnolia Drive, Tampa, FL 33612. Phone: 813-745-6484; Fax: Florida). LNCaP-Rf cells were provided by Dr. Donald Tindall (Mayo Clinic). 813-979-6700; E-mail: [email protected]. I2005 American Association for Cancer Research. LNCaP and DU-145 cells were cultured in RPMI 1640 containing 10% FCS. doi:10.1158/0008-5472.CAN-05-0347 PC3 cells were cultured in in DMEM containing 10% FCS. LNCaP-Rf cells www.aacrjournals.org 7717 Cancer Res 2005; 65: (17). September 1, 2005 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Cancer Research were cultured in RPMI 1640 containing 10% charcoal-stripped FCS. All homogenizer. The heavy membrane fraction was removed by two successive experiments were done on growing cells in medium containing 10% FCS. centrifugations, the first at 1,000 Â g for 10 minutes and the second at Stocks of roscovitine, R-roscovitine, and CGP74514A were prepared in 10,000 Â g for 10 minutes. To obtain the S100 fraction, the supernatant was DMSO. Roscovitine, LY294002, and pifithrin-a were purchased from centrifuged at 100,000 Â g for 90 minutes. Calbiochem (La Jolla, CA). R-roscovitine, z-DEVD-fmk, and z-LEHD-fmk Western blotting. Cells were rinsed with PBS and lysed in a buffer were obtained from Alexis Biochemicals (San Diego, CA). CGP74514A was containing 50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, purchased from Sigma (St. Louis, MO). Antibodies were obtained from Cell 1 mmol/L EGTA, 10% glycerol, 0.5% NP40, 1 mmol/L DTT, 0.1 mmol/L Signaling (Beverly, MA; Bad, PARP, and p53), Santa Cruz Biotechnology phenylmethylsulfonyl fluoride, 2.5 Ag/mL leupeptin, 0.5 mmol/L NaF, (Santa Cruz, CA; Bax), BD Transduction Laboratories (Lexington, KY; XIAP and 0.1 mmol/L Na3VO4. After a 30 minute incubation, insoluble material h and Bcl-XL), Alpha Diagnostic (San Antonio, TX; survivin), Sigma ( -actin was removed by centrifugation. Cell extracts normalized for amount and Flag), Upstate (Lake Placid, NY; Bak), Neomarker (Union City, CA; protein were resolved by SDS-PAGE and transferred to nitrocellulose p21Cip1), and Oncogene (Uniondale, NY; PUMA). membranes. Blots were blocked in PBS containing 0.05% Tween and 5% Keratin 18 cleavage. Immunocytochemistry using the M30 antibody to instant milk and incubated with antibody in PBS containing 0.05% Tween the keratin 18 cleavage product was done according to the instructions of for 2 hours at room temperature. Proteins recognized by the antibody were the manufacturer (Roche, Nutley, NJ). Twenty hours after plating in detected by enhanced chemiluminescence using a horseradish peroxidase– chamber slides, cells were incubated with roscovitine, R-roscovitine, coupled secondary antibody as specified by the manufacturer (Pierce, CGP74514A, or DMSO (vehicle control). After incubation, cells were fixed Rockford, IL). in ice-cold methanol at 20jC for 30 minutes and incubated with FITC- Preparation of adenoviruses encoding p53, XIAP, and XIAP small conjugated M30 antibody for 60 minutes at room temperature. Cells were interfering RNA. For preparation of XIAP small interfering RNA (siRNA), a mounted with Vectashield containing 4V,6-diamidino-2-phenylindole (DAPI). double-stranded oligonucleotide (5-GGCAGGTTGTAGATATATCAGCTC-V DNA fragmentation. DNA fragmentation was determined using a GAGCTGATATATCTACAACCTGCCCTTTTTG-3V)wassubclonedinto photometric enzyme-linked immunoassay (Cell Death Detection ELISA kit, pBluescript-U6 (provided by Dr. Yang Shi, Harvard University). The Roche) as specified by the manufacturer. Cells in 96-well plates were functionality of the resultant plasmid was ascertained by transient incubated in triplicate with roscovitine, R-roscovitine, CGP74514A, or transfection of 293 cells. pBluescript-U6 with and without insert was digested DMSO and lysed for 30 minutes at room temperature. Cell lysates were with XbaI to remove the U6 promoter (used for production of control virus) incubated with biotin-conjugated anti-histone antibody and peroxidase- and the U6 promoter plus the XIAP siRNA sequence. Excised DNA was conjugated anti-DNA antibody on streptavidin-coated microtiter plates for subcloned into the pAdTrack shuttle vector. Shuttle vectors were recombined 2 hours at room temperature. After incubation with ATBS substrate for with pAdEasy-1 according to the instructions of the manufacturer development of color, absorbance was read on a spectrophotometer at 405 (Stratagene, La Jolla, CA).