signaling pathway activated during apoptosis of the prostate cancer ... · prostate cancer remains...

[CANCER RESEARCH 59, 382–390, January 15, 1999]

Signaling Pathway Activated during Apoptosis of the Prostate Cancer Cell LineLNCaP: Overexpression of Caspase-7 as a New Gene Therapy Strategy forProstate Cancer1

Marco Marcelli, 2 Glenn R. Cunningham, Margaret Walkup, Zening He, Lydia Sturgis, Carolina Kagan,Roberta Mannucci, Ildo Nicoletti, BaBie Teng, and Larry DennerDepartments of Medicine [M. M., G. R. C., M. W., Z. H.] and Cell Biology [M. M., G. R. C.], Veterans Administration Medical Center and Baylor College of Medicine, Houston,Texas 77030; Institute of Internal Medicine and Oncologic Sciences, Perugia University Medical School, I-06100 Perugia, Italy [R. M., I. N.]; Institute of Molecular Medicine,University of Texas, Houston Health Science Center, Houston, Texas 77030 [BB. T.]; and Department of Cell Biology, Texas Biotechnology Corporation, Houston, Texas 77030[L. S., C. K., L. D.]

ABSTRACT

We studied the molecular mechanisms of apoptosis in the prostatecancer cell line LNCaP and whether overexpression of caspase activitycould force this cell line to undergo apoptosis. The inhibitor of phosphom-evalonate decarboxylase, sodium phenylacetate, and the protein kinaseinhibitor staurosporine induced (a) release of cytochromec from themitochondria to the cytosol; (b) reduction in mitochondrial transmem-brane potential; (c) proteolytic processing of caspase-3 and -7 but not -2;(d) cleavage of the DEVD substrate and the death substrates poly(ADP-ribose) polymerase and DNA fragmentation factor; and (e) apoptosis. Thepanspecific inhibitor of caspase activationN-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-FMK) prevented all of theseevents except release of mitochondrial cytochromec into the cytosol. Noneof these apoptotic signaling events were elicited by staurosporine orsodium phenylacetate treatment of LNCaP-Bcl-2 cells that overexpressthe oncoprotein Bcl-2. Because caspase-7 is activated in every model ofapoptosis that we have characterized thus far, we wished to learn whetheroverexpression of this protease could directly cause apoptosis of LNCaPcells. By using a replication-defective adenovirus, overexpression ofcaspase-7 protein in both LNCaP and LNCaP-Bcl-2 cells was accompa-nied by induction of cleavage of the DEVD substrate and TUNEL. Thesestudies have demonstrated that caspase-7 and -3 are critical mediators ofapoptosis in LNCaP cells. Caspase-7 was proteolytically activated in everymodel of apoptosis that we have developed, and the overexpression of itinduced apoptosis of LNCaP and LNCaP-Bcl-2 cells. Thus, adenoviral-mediated transfer of caspase-7 may offer a new effective approach for thetreatment of prostate cancer.

INTRODUCTION

Prostate cancer remains the second leading cause of cancer deathsin American men. The number of patients who died from this diseasein 1997 accounts for 14% of all cancer deaths in males (1). Radicalprostatectomy is the main curative treatment for men with organ-confined disease. Most men with non-organ-confined disease willundergo palliation with radiation or androgen ablation (2). Androgenablation successfully shrinks primary and metastatic lesions by induc-ing apoptosis or PCD3 of androgen-dependent prostate cancer cells

(3). Unfortunately, non-organ-confined prostate cancer is a heteroge-neous lesion and, at the time of diagnosis, contains foci of bothandrogen-dependent and -independent cells (4). Androgen-indepen-dent cells escape apoptosis induced by androgen ablation (5) and bymany cytotoxic drugs. They continue to proliferate and metastasizedespite profound changes in the surrounding hormonal milieu andrepresent the most direct threat to patient survival.

Despite its cryptic nature, the apoptotic machinery of prostatecancer cells is still in place (6) and can be activated under certaincircumstances (7). Therefore, we have hypothesized that the apoptoticmachinery of prostate cancer cells can be activated for tumoricidalpurposes and that an undesirable cell (e.g.,an androgen-independentprostate cancer cell) can be forced to commit PCD. LNCaP cells havean androgen receptor, and their growth is increased by androgen.However, because they do not undergo apoptosis after androgenwithdrawal, these cells can be used as anin vitro model to studystrategies for treating prostate cancers that are resistant to androgenablation. We previously described experiments in which the choles-terol-lowering medication lovastatin induced apoptosis of LNCaPcells (7) and demonstrated the involvement of the caspase family ofproteases in mediating apoptosis in this model. Caspase-7 was iden-tified as one of the possible mediators of lovastatin-induced apoptosis.

The goals of the current study were several (a) to identify thecaspase(s) activated in other models of apoptosis; (b) to determine themolecular steps of the apoptotic pathway that were induced upstreamand downstream of caspase activation; (c) to study the molecular basisof resistance to apoptosis in a LNCaP cell line stably transfected withthe oncoprotein Bcl-2; and (d) to determine whether overexpression ofa caspase activated during chemically induced PCD of LNCaP cellswould cause apoptosis of this cell line and of LNCaP-Bcl-2 cells inthe absence of apoptogenic stimuli. Here we present evidence further-ing our understanding of the molecular events associated with apop-tosis of LNCaP cells and demonstrate that the manipulation ofcaspase-7 is a potential gene therapy strategy to force prostate cancercells to undergo PCD.

MATERIALS AND METHODS

Materials. NPA was provided by Dr. W. Perkins (Elan PharmaceuticalResearch Corporation, Gainesville, GA). STS was purchased from SigmaChemical Co. (St. Louis, MO). Chemicals and protease inhibitors were pur-chased from Sigma and Boehringer (Indianapolis, IN). Fetal bovine serum andtissue culture media were from Life Technologies, Inc. (Gaithersburg, MD).The caspase inhibitors, z-VAD-FMK and z-DEVD-FMK, and the fluorogenicsubstrate, z-DEVD-AFC, were from Enzyme System (Dublin, CA). The en-

Received 7/29/98; accepted 11/11/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a Merit Review Grant from the Veterans Administration, by a PilotProject Grant from the Baylor Specialized Programs of Research Excellence on ProstateCancer, by a Grant from the Pfeiffer Foundation (to M. M.), and by a Grant fromAssociazione Italiana Picerca sul Cancro (AIRC) and Centro Universitario Ricerca On-cologica (CIRO) (to I. N.).

2 To whom requests for reprints should be addressed, at Department of Medicine,Baylor College of Medicine and Veterans Administration Medical Center, 2002 Hol-combe Boulevard, Houston, TX 77030. Phone: (713) 794-7945; Fax: (713) 794-7714;E-mail: [email protected].

3 The abbreviations used are: PCD, programmed cell death;Dcm, mitochondrialtransmembrane potential; NPA, sodium phenylacetate; STS, staurosporine; z-VAD-FMK, N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone; z-DEVD-FMK,N - benzyloxycarbonyl - Asp(OMe) - Glu(OMe) - Val - Asp(OMe) - fluoromethylketone;

Z-DEVD-AFC, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl cou-marin; JC-1, 5,59,6,69-tetrachloro-1,19,3,39-tetra-ethylbenzimidazolylcarbocyanine iodide,T-3168; TUNEL, terminal deoxynucletidyl transferase-mediated nick end labeling;PARP, poly(ADP-ribose) polymerase; DFF, DNA fragmentation factor; ICAD, inhibitorof caspase-activated DNase.

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hanced chemiluminescence detection reagents were from Amersham (Arling-ton Heights, IL). JC-1 was from Molecular Probes (Eugene, OR).

Cell Culture. LNCaP (obtained from the American Type Culture Collec-tion) were grown in RPMI 1640 supplemented with 10% fetal bovine serumand 1% penicillin and streptomycin. LNCaP-Bcl-2 (Ref. 8; gifts of Dr. R.Buttyan, Columbia University, New York, NY) were grown in RPMI 1640supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin,and 400mg/ml of Genetycin (Life Technologies).

STS and NPA dissolved in DMSO (Sigma) at concentrations 1000-foldhigher than the working concentrations were administered for various lengthsof time and doses (see “Results”). Controls received DMSO at a concentrationequal to that of the treated cells.

Apoptosis Assay.Apoptosis was determined by DNA laddering, TUNEL,and morphological criteria as described previously (7).

Confocal Microscopy. LNCaP and LNCaP-Bcl-2 cells were grown on25-mm glass coverslips in RPMI 1640 and 10% FCS. For mitochondrialanalysis, cells were loaded with 5mM JC-1 in RPMI 1640 for 10 min at 37°C.Cells were then washed twice with PBS and transferred to an Attofluormicrochamber (Molecular Probes) for confocal analysis on a MCR-1024Bio-Rad apparatus. A representative microscopic field was chosen and abaseline image was recorded. The 488-nm laser line was used for excitation,and red and green emissions of the sample were simultaneously recorded. Themicroscopic field, laser potency, and photomultiplier gain were kept constantfor the whole experiment. Images were processed with the LaserSharp 1024(Bio-Rad) and reanalyzed with Optilab-Pro softwares (Graftek Imaging, Aus-tin, TX) on a PowerPC personal computer (MacIntosh 8100, Apple, Cupertino,CA). STS (4mM) was then added and images were registered after 10, 20, 30,40, and 60 min. For the 4-h experiments, cells were grown on coverslips asdescribed above and incubated with 4mM STS for 4 h. When the pancaspaseinhibitor was used, cells were pretreated for 1 h with 100mM z-VAD-FMK.After a 4-h incubation, cells were loaded with 5mM JC-1 for 10 min at 37°Cin culture medium. Cells were then washed twice with PBS, the coverslip wasmounted onto the Attofluor chamber, and confocal analysis was performed asdescribed above.

Western Analysis. The techniques and the antibodies for the immunode-tection of PARP, caspase-3, and caspase-7 have been described previously (7).For the immunodetection of caspase-2 (Transduction Laboratories, Lexington,KY) and DFF (antibody provided by Dr. Xiaodong Wang, University of TexasSouthwestern Medical Center, Dallas, TX), we used the same technique usedfor caspase-3 and -7. Cytosolic extracts free of mitochondria were prepared forthe immunodetection of cytochromec as described by Bossy-Wetzelet al. (9).Briefly, LNCaP cells grown in 10-cm dishes were collected by microcentrifu-gation at 1500 rpm at 4°C for 2 min and washed twice in ice-cold PBS. Thecell pellet was resuspended in 200ml of extraction buffer [220 mM mannitol,68 mM sucrose, 50 mM PIPES-KOH (pH 7.4), 50 mM KCl, 5 mM EGTA, 2 mM

MgCl2, 1 mM DTT, and protease inhibitors (complete™, Boehringer, Indian-apolis IN)]. After 30 min at 4°C, cells were homogenized with a glass dounceand a B pestle (40 strokes). The homogenates were spun at 13,000 rpm for 15min, and the supernatants were stored at270°C. Cytosolic protein extracts (50mg) were then sized in 17% SDS-polyacrylamamide gel, blotted, and analyzedusing monoclonal antibody 7H8.2C12 (PharMingen, San Diego, CA). Immu-noreactive bands were visualized using the enhanced chemiluminescencedetection reagents from Amersham Corp. (Arlington Heights, IL).

Assay of DEVDase Activity. The catalytic activity of the caspases wasmeasured as described previously (7) using a fluorometric assay monitoringthe cleavage of the fluorogenic tetrapeptide z-DEVD-AFC.

Inhibition of Caspase Activity. Inhibition of caspases with DEVD cleav-age activity was achievedin vivo using the inhibitors z-DEVD-FMK andz-VAD-FMK administered at 100mM 1 h before the addition of STS (4mM).Twelve h after the addition of STS, cells were harvested and analyzed byTUNEL, Western analysis, or DNA electrophoresis.

Preparation of Adenovirus. A caspase-7 cDNA encompassing amino acids1–303 (10) of caspase-7 was obtained by reverse-transcription PCR of LNCaPcells mRNA. The product of the amplification was subcloned in theBamHI andXbaI sites of plasmid pCDNA-3 (Invitrogen, Carlsbad CA). The absence ofmutations in the resulting construct was evaluated by sequence analysis. Replica-tion-defective recombinant adenoviruses were produced as reported previously(11). Briefly, the caspase-7 cDNA was subcloned in vector pAvS6 (12) to obtainthe shuttle plasmid pAvS6–C7. pAvS6 has a pBluescript (Stratagene) backbone

and contains sequences from the left end of the adenovirus 5 genome lacking theearly transcription region-1 (E1) that is necessary for replication. In this plasmid,the gene of interest is subcloned in a polylinker site located downstream from theRSV promoter and upstream to the SV40 early polyadenylation signal. pAvS6–C7was cotransfected with pJM17 (13), which contains a full-length adenoviralgenome, in low-passage 293 cells (14). This cell line is stably transfected with theSV40 virus and provides E1 functions in trans (Microbix Biosystem, Toronto,Canada). E1-defective recombinant adenovirus (Av-C7) was produced by homol-ogous recombination between pAvS6–C7 and pJM17 in 293 cells. Two weeksafter transfection, infectious recombinant adenoviral plaques were picked, ex-panded, and screened for caspase-7 sequences by PCR. Adenoviral vectors thatcontained C7 were purified one more time by plaque assay on 293 cells, andrecombinant Av-C7 was confirmed by sequencing and restriction-enzyme map-ping. Recombinant adenovirus Av–lacZ, which contains the lacZ cDNA under thecontrol of the RSV promoter, was produced as described previously (11). Large-scale production of high-titer recombinant adenovirus was performed by growing293 cells on improved Eagle’s MEM supplemented with 10% fetal bovine serum,2 mM L-glutamine, 50 units/ml penicillin, 50mg/ml streptomycin, and 1% Fun-gizone (Life Technologies). The virus was purified twice using cesium chloridedensity gradient centrifugation. The viral vector was then dyalyzed for$8 h at 4°Cagainst a buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, and 10%glycerol and was stored at280°C.

Infection of LNCaP Cells with Viral Constructs. In preliminary experi-ments, we titrated the concentration of Av-C7 that achieved apoptosis ofLNCaP cells in the most effective and specific way. Experiments were per-formed in which 43 105 LNCaP cells (seeded in 6-well plates) were infectedwith 108-1011 viral particles of Av-C7, the control virus Av-lacZ, or vehiclealone. At the time points of 24, 48, 72, and 96 h, cells were harvested andanalyzed for TUNEL. Because the apoptogenic activity of Ad-C7 was mosteffective at the titer of 1010, LNCaP and LNCaP-Bcl-2 cells were treated with1010 viral particles of Av-C7 or Av-lacZ for 0–96 h. At the time points of 24,48, 72, and 96 h, cells were harvested and analyzed for caspase-7 expressionand processing, TUNEL, and DEVD-ase activity.

RESULTS

Apoptosis of LNCaP Cells.To determine whether NPA and STSinduce the apoptosis of LNCaP cells, LNCaP cells were incubatedwith increasing concentrations of NPA (0.1–10 mM for 96 h) and STS(4 nM–40 mM for 24 h; data not shown). The minimal concentrationthat induced reproducible apoptosis assessed by DNA laddering wasused for subsequent experiments. Fig. 1 shows the kinetics of STS-(1A) and NPA- (1B) induced apoptosis. Treatment with NPA (10 mM)was associated with induction of apoptosis after 48 h of treatment.STS (4mM) was considerably more rapid and induced apoptosis by6 h. After 24 h of treatment with STS, 876 4% of the cells wereTUNEL-positive, in comparison with 4.26 0.4% of the cells receiv-ing vehicle alone (Table 1). After 72 h of treatment with NPA,80 6 6% of the cells were TUNEL-positive, in comparison with6 6 0.98% of the cells receiving vehicle (Table 1).

To evaluate mitochondrial involvement, we induced apoptosis withSTS and used confocal microscopy of LNCaP cells stained with thefluorescent cyanine dye JC-1 (Fig. 2), which is specifically taken upby the mitochondria. When the transmembrane potential (Dcm) of themitochondria is high, like it is in a normal cell, JC-1 forms redfluorescent dimers. However, when theDcm is low, as in mitochon-dria of cells undergoing apoptosis, the red fluorescence disappears.After treatment with STS for 1 (data not shown) or 4 h (Fig. 2,A andB), the red mitochondrial fluorescence disappeared, which indicates adecrease inDcm concomitant with the induction of apoptosis. Othermorphological changes of LNCaP cells undergoing apoptosis in-cluded ballooning of the mitochondria (data not shown).

Apoptosis of LNCaP-Bcl-2 Cells.The ability of STS (4mM) toinduce apoptosis of LNCaP-Bcl-2 cells also was determined. DNAladdering (Fig. 1C) and TUNEL (2.36 0.2%versus3.26 0.1% cellswere TUNEL-positive after 24 h in the STS and control group,

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APOPTOSIS IN PROSTATE CANCER CELL LINES

respectively; Table 1) demonstrated that LNCaP-Bcl-2 cells did notundergo apoptosis when exposed to STS. LNCaP-Neo cells (trans-fected with an empty vector and functioning as control for LNCaP-Bcl-2) underwent apoptosis with the same kinetics as native LNCaPcells (data not shown). In addition, no changes in the fluorescence ofJC-1 stained mitochondria was visible (Fig. 2,D and E), indicatingthat Bcl-2 overexpression had a protective effect on theDcm of thiscell line. Similarly, NPA was unable to induce apoptosis of LNCaP-Bcl-2 cells (Table 1).

Release of Mitochondrial Cytochromec during STS- and NPA-induced Apoptosis.We hypothesized that mitochondrial factorswere released into the cytosol during STS- and NPA-induced apop-tosis, as described in other models of apoptosis (15, 16). Therefore,we sought to determine whether cytochromec, an intramitochondrialprotein in nonapoptotic cells, was released in the cytosol of LNCaPcells in our models of apoptosis. As the apoptosis of LNCaP cellsproceeded, cytochromec accumulated in the cytosol. This event wasevident after 1 h and peaked after 6 and 12 h of STS treatment (Fig.3A) and after 24 h of NPA treatment (Fig. 3B).

Induction of DEVDase Activity. Caspase activation was deter-mined by incubating the fluorogenic peptide Ac-DEVD-AFC withcell lysates of LNCaP cells undergoing PCD during treatment withSTS or NPA. DEVDase activity, which indicates activation of groupII caspases (17), was induced 2.446 0.06- and 1.246 0.84-fold after12 h of STS and after 48 h of NPA stimulation, respectively.

Proteolytic Processing of Caspase-2, -3, and -7.On the basis ofthe results described above, immunoblot analyses of lysates obtainedfrom LNCaP cells undergoing apoptosis during treatment with NPAand STS was performed using antibodies for the currently knownmembers of group II caspases (i.e.,caspase-2, -3, and -7; Ref. 17). Wefound that caspases-3 (Fig. 4,B andE) and -7 (Fig. 4,C andF) butnot caspase-2 (Fig. 4,A andD) were proteolytically cleaved in LNCaPcells undergoing apoptosis during treatment with STS and NPA.

Although the experiments shown in Fig. 4 suggest that caspase-7 wascleaved before caspase-3 (theMr 20,000 active subunit of caspase-7appeared after 1 h of STS and 24 h of NPAtreatment), this maysimply be related to differences in the ability of the antibodies thatwere used to detect the active subunits or in the stability of the activesubunits of caspase-3 and -7.

Because LNCaP-Bcl-2 cells failed to undergo apoptosis whentreated with STS, we investigated whether this was associated with thefailure to activate caspase-2, -3, and -7. The experiments presented inFig. 5, A-C show that no proteolytic cleavage of these caspases wasdetectable in cell lysates obtained after 1, 3, 6, 12, and 24 h oftreatment with 4mM STS. Similar results were also obtained aftertreatment with NPA (data not shown).

Cleavage of PARP and DFF.An increasing number of down-stream targets of caspase-3 and -7 have been identified. We focusedour attention on PARP, a repair enzyme that is cleaved and inactivatedby caspase-3 and -7 (18), and DFF (19) or ICAD (20, 21), thecleavage of which is necessary for the activation of the caspase-activated DNase (CAD; Ref. 20) that is responsible for internucleo-somal DNA degradation during apoptosis. Treatment with NPA andSTS was associated with cleavage of the substrates PARP and DFF/ICAD (Fig. 6). In LNCaP-Bcl-2 cells, in which STS (Fig. 1C) andNPA (data not shown) did not induce apoptosis, no cleavage of PARPor DFF/ICAD (Fig. 5,D andE) was observed.

Inhibition of Caspase-3 and -7 Activation. To further linkcaspase-3 and -7 activation to apoptosis of LNCaP cells, cells weretreated for 12 h with STS6 the caspase inhibitors z-VAD-FMK orz-DEVD-FMK. Although treatment with the panspecific z-VAD-FMK completely inhibited STS-induced DNA laddering(Fig. 7A)and TUNEL positivity (STS-treated cells: 456 3% TUNEL-positive cells; STS- and z-VAD-treated cells: 86 1.6% TUNEL-positive cells; vehicle-treated cells: 4.16 0.3% TUNEL-positivecells), treatment with the more narrowly specific z-DEVD-FMKminimally inhibited DNA laddering (Fig. 7A) and TUNEL (STS-and z-DEVD-treated cells: 376 4% TUNEL-positive cells; STS-treated cells: 456 3% TUNEL-positive cells). The different abilityof the two inhibitors to prevent apoptosis was probably due to theirdifferent effectiveness in preventing caspase-3 and -7 activation.This is shown in the Western analysis of caspase-3, -7, DFF, andPARP obtained from lysates of LNCaP cells treated for 12 h withSTS 6 z-VAD-FMK or z-DEVD-FMK (Fig. 7, B-E). Althoughz-VAD-FMK completely prevented the processing of caspase-3and -7, as well as the cleavage of the two substrates DFF andPARP, z-DEVD-FMK was only partially effective in this regard(Fig. 7,B-E). These experiments showed conclusively that preven-tion of apoptosis occurred only when caspase-3 and -7 activationwas completely inhibited.

Mechanisms of Apoptosis Inhibition. Because the accumulationof cytochromec in the cytosol is a fundamental event mediating theprogression of apoptosis in other systems (15, 16), we investigatedwhether thr inhibition of STS-induced apoptosis with z-VAD-FMK or

Fig. 1. STS and NPA induce apoptosis of the cell line LNCaP but not of LNCaP-Bcl-2cells. Agarose electrophoresis of LNCaP cells treated with STS (4mM) for 0–24 h (A) orwith NPA (10 mM) for 0–72 h (B). C shows LNCaP-Bcl-2 cells treated with STS (4mM)for 0–24 h.MW, molecular weight markers.

Table 1. Percent of TUNEL positive LNCaP and LNCaP-Bcl-2 cells (mean6 SD) during treatment with vehicle, STS, and NPA

Hours

0 1 3 6 12 24 48 72

LNCaPVehicle 2.66 0.3 3.16 0.05 2.56 0.7 3.76 0.3 46 0.44 4.26 0.4 5.36 1 66 0.98STS 36 1 66 0.5 136 2.34 356 7 706 11 876 4NPA 1.56 0.5 10.56 1.9 44.56 7 806 6

LNCaP-Bclp2Vehicle 1.56 0.4 16 0.5 1.46 0.3 1.76 0.8 2.56 0.7 3.26 0.1 3.56 1 56 0.87STS 1.56 0.8 0.46 0.01 1.36 0.4 26 0.4 1.46 0.1 2.36 0.2NPA 0.46 0.02 3.16 0.3 4.16 0.76 66 1

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Bcl-2 overexpression was associated with alterations in the intracel-lular distribution of cytochromec, and in the prevention ofDcm

changes. Fig. 2C shows that z-VAD-FMK, similarly to Bcl-2 over-expression (Fig. 2E), prevented changes of the transmembrane poten-tial of LNCaP cells treated with STS for 4 h. However, z-VAD-FMKwas unable to prevent STS-induced cytochromec translocation intothe cytosol, whereas this phenomenon was successfully inhibited byBcl-2 overexpression (Fig. 8,A and B, respectively). These experi-ments demonstrated that Bcl-2 overexpression or treatment with z-VAD-FMK prevented STS-induced apoptosis with different mecha-nisms of action. Bcl-2 overexpression blocked cytochromectranslocation and subsequent caspase activation, whereas z-VAD-FMK worked further downstream by direct inhibition of caspaseactivation.

Apoptosis Induced by Viral-mediated Overexpression ofCaspase-7 in LNCaP and LNCaP-Bcl-2 Cells.Since caspase-7 isactivated in every model of apoptosis that we have characterizedthus far, we wished to learn whether overexpression of this prote-ase could directly cause apoptosis of LNCaP cells. These experi-ments were done by infecting LNCaP cells with Av-C7 or Av-lacZ.The resulting transiently infected cells were named LNCaP-C7 and

LNCaP-lacZ. Cells were harvested at 24, 48, 72, and 96 h andanalyzed for caspase-7 expression and processing, DEVDase ac-tivity, and TUNEL. Induction of caspase-7 protein overexpressionand processing was present only in cells infected with Ad-C7 (Fig.9B), and was evident at 48 h postinfection. There was a 7.5-foldinduction of DEVDase activity that became evident after 48 h andremained constant throughout the experiment in cells infected withAd-C7 (Fig. 9A). The percentage of apoptotic cells was15.5 6 0.7% and 806 3% after 72 and 96 h postinfection,respectively, in LNCaP-C7. There was a 1.86 0.2% and a6.5 6 1% background apoptotic index in LNCaP-lacZ cells at 72and 96 h, respectively (Fig. 9C).

Similar experiments were performed with the cell line LNCaP-Bcl-2, and the resulting cell lines were named LNCaP-Bcl-2-C7 andLNCaP-Bcl-2-lacZ. Again, infection with Av-C7 was specificallyassociated with the induction of caspase-7 protein expression andprocessing (Fig. 10B), DEVDase activity (5.5-fold above LNCaP-Bcl-2-lacZ; Fig. 10A), and TUNEL (Fig. 10C). Overexpression ofcaspase-7 peaked at 72 h postinfection and was 45-fold higher than inLNCaP-Bcl-2-lacZ (Fig. 10B). The apoptotic index in LNCaP-Bcl-2-C7 cells was 386 2% at 96 h and was 3-fold higher than LNCaP-Bcl-2-lacZ cells (Fig. 10C).

DISCUSSION

Proteolytic Activation of Caspase-3 and -7 Is a Common EventLeading to Apoptosis of LNCaP Cells.Two substances with differ-ent mechanisms of action—NPA (a phosphomevalonate inhibitor) andSTS (an inhibitor of several protein kinases and topoisomerase II)—induced the apoptosis of LNCaP cells by similar signaling pathways.

Fig. 2. Reduction inDcm in LNCaP cells after treatment with STS and prevention by overexpression of Bcl-2 or treatment with the caspase inhibitor z-VAD-FMK. LNCaP (A-C)or LNCaP-Bcl-2 cells (C-E) were treated with vehicle (A, C), STS [4mM for 4 h (B, E)], or STS1z-VAD-FMK [4 and 100mM, respectively, for 4 h (C, F)]. After 4 h, cells were loadedwith JC-1 (5mM) for 10 min. Cells were then analyzed on a confocal microscope as described in “Materials and Methods.”Red fluorescence, negativeDcm; N, nucleus.

Fig. 3. Cytochromec accumulation in the cytosol during STS- and NPA-inducedapoptosis of LNCaP cells. Immunoblot analysis of cytochromec in cytosolic lysatesobtained from LNCaP cells treated with STS (4mM) for 0–12 h (A) or with NPA (10 mM)for 0–72 h (B).

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Shortly after treatment with these agents, we observed the release ofcytochromec from the mitochondria to the cytosol, activation ofcaspase-3 and -7, and cleavage of the downstream targets of activatedcaspases, PARP and DFF/ICAD. When the pan-caspase inhibitorz-VAD-FMK was used to prevent caspase-3 and -7 activation, STSand NPA were unable to induce apoptosis of LNCaP cells andcleavage of the downstream targets PARP and DFF/ICAD. A similarscenario was observed in LNCaP-Bcl-2 cells, in which resistance toSTS and NPA-induced apoptosis was associated with the lack ofcaspase-3 and -7 activation or PARP and DFF/ICAD cleavage. Theseobservations, together with a previous report in which we observedthat lovastatin induces apoptosis of LNCaP cells by activatingcaspase-7 (7), have delineated the central role played by the caspases(in particular caspase-7) in mediating apoptosis of LNCaP cells. Themechanism through which STS and NPA activate the apoptotic path-way is still undetermined. Interestingly, a lower dose of STS inhibited12-O-tetradecanoylphorbol-13-acetate (TPA)-induced apoptosis ofLNCaP cells by interfering with the protein kinase C pathway (22).The reason why we (and many others) have observed induction ofapoptosis by STS is probably related to additional effects that STSmay have when used at 80-fold higher concentration [i.e., inhibition ofother protein kinases (23) and/or topoisomerase II (24) activity].

z-VAD-FMK and Bcl-2 Overexpression Prevent Apoptosis withDifferent Mechanisms of Action. Translocation of cytochromecinto the cytosol is one of the first events occurring in many cells

undergoing apoptosis (9, 25). This phenomenon is followed by theproteolytic activation of the early caspases in the apoptosome (26), acomplex of apoptogenic factors consisting of the mammalian CED-4homologue APAF-1 (27), caspase-9 (28, 29), cytochromec, anddATP (30). It has been suggested that during apoptosis, cytochromecaccumulates into the cytosol in which it binds Apaf-1 and inducesconformational changes that allow Apaf-1 to bind caspase-9, which inturn is cleaved into its active subunits (30, 31). dATP is thought toprovide the energy necessary for the activation of caspase-9 (30).Caspase-9, or other cell type-specific upstream caspases, is the earliestcaspase activated in nonreceptor-mediated apoptosis (30). Activecaspase-9 then cleaves and activates other downstream caspases,including caspase-3 and -7, which sets in motion the execution phaseof apoptosis (30).

In LNCaP cells, apoptosis induced by STS or NPA was averted byz-VAD-FMK treatment or Bcl-2 overexpression. Although both meth-ods of inhibition prevented activation of caspase-3 and -7, the mech-anisms were different. Bcl-2 prevented translocation of cytochromecto the cytosol and, consequently, the activation of the early caspase(s)that at this time are unidentified for LNCaP cells. z-VAD-FMKdirectly prevented the proteolytic activation of the caspases (32) butdid not prevent the translocation of cytochromec to the cytosol.Notably, both Bcl-2 overexpression and treatment with z-VAD-FMKprevented changes in theDcm. These observations agree with some(9) but not other reports (33) and imply that cytochromec transloca-

Fig. 4. Activation of caspase-3 and -7 duringSTS- and NPA-induced apoptosis of LNCaP cells.Immunoblot analysis of caspase-2 (A andD), -3 (Band E) and -7 (C and F) in LNCaP cell lysatesobtained during treatment with STS (4mM, 0–24 h,A-C), or NPA (10 mM, 0–72 h,D-F). Activation ofcaspase-3 is indicated by the disappearance of theprecursor band; activation of caspase-7 is indicatedby the progressive disappearance of the precursorband and by the appearance of theMr 20,000 (20kDa) active subunit.

Fig. 5. LNCaP-Bcl-2 cells resist STS-induced ap-optosis. Immunoblot analysis of caspase-2 (A), -3(B), -7 (C), DFF (D), and PARP (E) in LNCaP-Bcl-2 cell lysates obtained during treatment withSTS (4mM, 0–24 h).

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tion does not require a mitochondrial transmembrane depolarizationand that the decrease of theDcm is a consequence of caspase activa-tion. An important conclusion derived from these experiments is thatthe prevention of caspase activation is a mechanism-independentevent necessary for the prevention of apoptosis in LNCaP cells andpresumably in other prostate cancer cells.

Bcl-2 Is a Global Inhibitor of Apoptosis That Is Overexpressedin Prostate Cancer. Prostate cancer arises from the secretory cells ofprostatic epithelium, and Bcl-2 is a well characterized antiapoptoticfactor that may play an important role in this disease. In benignprostatic epithelium, Bcl-2 is expressed in the basal cell layer but notin the secretory cells (34–36). The pattern of Bcl-2 expression inprostate cancer varies with disease progression. Before androgenablation, Bcl-2 is not expressed or is heterogeneously expressed (36,37) in many patients with prostate cancer. However, in a subset ofsuch patients, Bcl-2 is present and predicts poor survival (38–40). Inprimary or metastatic specimens obtained from individuals failingandrogen ablation, Bcl-2 is frequently overexpressed (36, 37, 41).Thus, these observations identify Bcl-2 expression as a possibleprognostic risk for patients with prostate cancer. Two mechanismscould explain the accumulation of cells expressing this oncoprotein.Bcl-2 expression could be stimulated by androgen ablation so that the

Fig. 6. Cleavage of PARP and DFF in LNCaPcells undergoing apoptosis after treatment withSTS or NPA. Immunoblot analysis of PARP (AandC) and DFF (B andD) in LNCaP cell lysatesobtained during treatment with STS [4mM, 0–24 h(A andB)] or NPA [10 mM, 0–72 h (C andD)]. Theprocessing of PARP is indicated by the progressivedisappearance of theMr 115,000 (115 kDa) full-length protein and by the progressive appearanceof the Mr 85,000 (85 kDa) cleaved fragment.Cleavage of DFF is indicated by the progressivedisappearance of theMr 45,000 (45 kDa) and Mr

40,000 (40 kDa) subunits. The migration of thefragments of PARP and DFF differs from that inFig. 4 because two different gel apparatuses wereused in the two experiments (Figs. 4 and 6), whichaccounts for a longer migration in the experimentshown in Fig. 6.

Fig. 7. Prevention of STS-induced apoptosis of LNCaP cells is elicited by the caspaseinhibitor z-VAD-FMK but not z-DEVD-FMK.A, agarose gel electrophoresis of genomicDNA extracted from LNCaP cells 12 h after treatment with vehicle alone (Control), STS(4 mM) 1 z-VAD-FMK (100 mM), STS (4mM) 1 z-DEVD-FMK (100 mM), or STS (4mM). MW, molecular weight markers.B–E, immunoblot analysis of caspase-3, -7, DFF,and PARP, respectively, in LNCaP cell lysates after 12 h of treatment with vehicle alone(Control), STS1z-DEVD-FMK (4 and 100mM), STS1z-VAD-FMK (4 and 100mM), andSTS alone (4mM). Processing of caspase-3, DFF, and PARP is indicated by the disap-pearance of the major band. Processing of caspase-7 is indicated by the disappearance ofthe major band and by the appearance of theMr 20,000 (20kDa) subunit. InC, there is aMr 20,000 (20kDa) band also in the cells treated with vehicle alone, which indicates somebaseline activation of caspase-7. When STS was given alone, the intensity of theMr

20,000 (20kDa) band increased significantly, which indicates an accumulation of theactive form of caspase-7 after treatment with STS.

Fig. 8. Cytochromec translocation to the cytosol is inhibited by Bcl-2 overexpressionbut not by treatment with z-VAD-FMK.A, immunoblot analysis of cytochromec incytosolic lysates from LNCaP cells extracted after 12 h of treatment with vehicle alone,STS (4 mM), or STS1z-VAD-FMK (4 and 100 mM, respectively).B, cytochromecexpression in cytosolic lysates from LNCaP-Bcl-2 cells after treatment with vehicle aloneor STS (4mM for 12 and 24 h).

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tissue can resist androgen ablation-induced apoptosis. Such a possi-bility is suggested by the observation that castration up-regulates theexpression of prostatic Bcl-2 mRNA in mice (37) and of Bcl-2immunoreactivity in prostatic tissue obtained before and after andro-gen ablation in patients with prostate cancer (42) or benign prostatichyperplasia (35). Alternatively, it could be that cells already overex-pressing Bcl-2 are selected by androgen ablation, in part, for theirability to resist apoptosis and, in part, for their increased malignantphenotype. Such possibilities are suggested by the observation thatBcl-2 overexpression in LNCaP cells confers an androgen-indepen-dent (8) and a metastatic (43) phenotype. Down-regulation of Bcl-2expression may have therapeutic benefits in some circumstances. Forinstance, antisense oligos for this oncoprotein have been described todecrease its expression and reconstitute sensitivity to etoposide-induced apoptosis in LNCaP cells (44).

Our studies with LNCaP-Bcl-2 cells have been helpful in under-standing the molecular bases of cell-death resistance in prostate can-cers overexpressing this oncoprotein and have shown that Bcl-2overexpression prevents the activation of caspase-3 and -7 while alsopreventing the accumulation of cytochromec in the cytosol. Becausecaspase activation occurs downstream from the Bcl-2 check point(45), this observation has clinical implications and suggests thatactivation of the caspases can induce apoptosis in prostate cancerscells that overexpress Bcl-2.

Overexpression of Caspase-7 Induces Apoptosis of LNCaP andLNCaP-Bcl-2 Cells. The observation that STS-induced apoptosis ofLNCaP cells occurs if caspase-3 and -7 are activated implies that thesecaspases are potential therapeutic targets to repress or to induceapoptosis. Manipulation of the apoptotic pathway for the treatment ofhuman diseases is becoming an important field of investigation inas-much as many conditions associated with excess or impaired PCDhave been described (46).

Prevention or enhancement of caspase activity for therapeutic pur-

poses has already been demonstrated in many animal disease models.The damage observed in the central nervous system of animals un-dergoing cerebral ischemia can be minimized by using inhibitors ofcaspase activation in an acute setting (47–49). Similar results havebeen obtained in animal models of miocardial ischemia (50, 51). Yuet al. (52) induced therapeutic apoptosis of a rat glioma model usinga retrovirus containing the cDNA of caspase-1. Kondoet al. (53)successfully induced apoptosis of human and murine malignant gli-oma cellsin vivo andin vitro after retroviral transfer of caspase-3 and-6. Additional evidence supports the potential tumoricidal ability ofthe caspases. On transfection and subsequent overexpression, many ofthese proteases can induce apoptosis of the host cell (28, 54–56).Other caspases, including caspase-7, require the removal of the NH2-terminal prodomain to effectively induce apoptosis of the host cell(10, 57, 58).

Because caspase-7 was an active participant in every model ofapoptosis of LNCaP cells that we have developed thus far and theprevention of its activation by z-VAD-FMK or Bcl-2 was associatedwith the prevention of apoptosis, we hypothesized that LNCaP andLNCaP-Bcl-2 cells would undergo apoptosis after the overexpressionof this protease. A caspase-7 cDNA was incorporated into a firstgeneration adenoviral vector in which the expression of the gene ofinterest is under the control of the powerful RSV promoter. Overex-pression of caspase-7 protein paralleled by a concomitant increase ofDEVD-ase activity was seen after 48 h, and apoptosis was evident by72 h postinfection. Importantly, overexpression of caspase-7 was ableto bypass the antiapoptotic activity of the oncoprotein Bcl-2 in the cellline LNCaP-Bcl-2.

These experiments have several implications. They have demon-strated that the naturally occurring caspase pathway can be manipu-lated in anin vitro setting to induce apoptosis of a prostate cancer cellline generated from a patient affected by androgen-independent pros-tate cancer. More importantly, it seems that caspase overexpression

Fig. 9. Adenoviral-mediated overexpression ofcaspase-7 induces apoptosis of LNCaP cells. DEV-Dase activity (A), caspase-7 expression (B), and %TUNEL-positive cells (C) detected in LNCaP-C7and LNCaP-lacZ cells at time points 24–96 hpostinfection with 1010 particles of Av-C7 and Av-lacZ, respectively.

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has the ability to bypass the antiapoptotic effect of the oncoproteinBcl-2, a putative mediator of apoptosis resistance in androgen-inde-pendent prostate cancer.

The observations described in this paper provide the theoreticalfoundation for a new and promising therapeutic approach for prostatecancer. Adenovirus-mediated overexpression of caspase-7 may be auseful gene therapy approach to treat advanced prostate cancer. Itremains to be seen whether caspase overexpression is a feasibleapproach to induce apoptosisin vivo, where the growth of prostaticepithelium is under the physiological stimulation of endocrine andparacrine growth factors and where the anatomical boundaries withthe adjacent structures (i.e., the prostatic stroma) are intact. Futurestudies will address these issues as well as the need to limit caspase(over)expression to prostatic epithelium by using tissue-specific pro-moters.

ACKNOWLEDGMENTS

We thank Drs. Buttyan (Columbia University, New York, NY), Wang(University of Texas Southwestern Medical Center, Dallas, TX), and Dixit(Genentech, San Francisco, CA) for reagents. NPA was a gift from Dr. W.Perkins, Elan Pharmaceutical Research Corporation (Gainesville, GA).

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signaling pathway activated during apoptosis of the prostate cancer ... · prostate cancer remains...

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