loss of caspase-1 and caspase-3 protein …...caspase-3 is the ultimate executioner caspase that is...

[CANCER RESEARCH 61, 1227–1232, February 1, 2001]

Loss of Caspase-1 and Caspase-3 Protein Expression in Human Prostate Cancer1

Rachel N. Winter, Andrew Kramer, Andrew Borkowski, and Natasha Kyprianou2

Departments of Biochemistry and Molecular Biology [R. N. W., N. K.], Urologic Surgery [A. K., N. K.], and Pathology [A. B.], University of Maryland School of Medicine,Baltimore, Maryland 21201

ABSTRACT

Activation of the caspase cascade is involved in the execution of apo-ptosis in a variety of cellular systems. Recent studies demonstrated thatcaspase-1 activation was required for human prostate cancer cells toundergo apoptosis in response to transforming growth factor-b (Y. Guoand N. Kyprianou, Cancer Res.,59: 1366–1371, 1999). In the presentstudy, to identify the significance of caspases in prostate cancer progres-sion, we examined the expression of three key caspases, caspase-1,caspase-3, and caspase-9, in normal and malignant human prostates.Caspase-1, -3, and -9 expression was examined at the mRNA and theprotein level in a series of human normal and malignant prostate speci-mens. No significant differences were observed in the mRNA expression inprostatic tumors relative to the normal gland for any of the three caspases.Immunohistochemical analysis revealed that the pattern of protein ex-pression and distribution was uniformly homogeneous in the normalprostate, with the epithelial cells exhibiting a diffuse cytoplasmic stainingfor caspase-1 and caspase-3. Significantly, the majority of primary pros-tate cancer specimens (80%) had total lack of caspase-1 immunoreactivity,whereas the remaining showed a significantly reduced expression com-pared with the normal prostate (P < 0.05). Caspase-3 expression was alsoreduced in moderately and poorly differentiated prostatic tumors com-pared with well-differentiated prostate adenocarcinomas and the normalprostate (P < 0.05). No significant correlation was found between theapoptotic index or Gleason grade and the pattern of caspase proteinexpression in the primary prostatic tumors analyzed. Western blot anal-ysis revealed constitutive expression of the proenzyme forms of caspase-1,-3, and -9 in the human prostate cancer cell lines PC-3, DU-145, TSU-Pr1m and LNCaP, but caspase-1 expression was low in the less tumori-genic cell lines, DU-145 and LNCaP. These findings implicate the loss ofcaspase-1 protein as a potential step in the loss of apoptotic control duringprostate tumorigenesis. This study suggests that the pattern of caspase-1and -3 expression in prostatic tumors may have prognostic significance indisease progression.

INTRODUCTION

Prostate cancer is the second highest cause of cancer death in malesafter lung cancer. As the age of the American population increases, anestimated 178,300 American men will be diagnosed with prostatecancer in 2000. The probability of developing prostate cancer is 1 in55 in males ages 40–59 and 1 in 6 in males ages 60–79 (1). Prostatecancer mortality results from metastasis to the bone and lymph nodesand progression from androgen-dependent to androgen-independentprostatic growth (2). Prostate cancer patients, although initially re-sponsive to hormone-ablation therapy, often relapse with androgen-independent disease that is resistant to further therapeutic interventions.

Apoptosis is the physiologically relevant mode of programmed celldeath that counterbalances cell proliferation (3). Tissue homeostasis inthe normal prostate gland is maintained by the quantitative relation-ship between the rate of cell proliferation and the rate of apoptotic cell

death (4). Recent kinetic studies on the dynamics of prostate growthby this laboratory and other investigators suggest that disruption of themolecular mechanisms that regulate apoptosis and cell proliferationamong the epithelial cells is responsible for the abnormal growth ofthe gland during neoplastic development (5). Identification and tar-geting of cellular modifiers of apoptosis for implementing effectivetherapeutic strategies for advanced prostate cancer has been the focusof intense efforts.

The mechanism of apoptosis is remarkably conserved across spe-cies, and is executed with a cascade of sequential activation ofinitiator and effector caspases (6). Caspases, members of the cysteineprotease family, are synthesized as inactive proenzymes and areselectively cleaved after an aspartate residue to produce the activeenzyme (7, 8). Once activated, the effector caspases can cleave abroad range of cellular targets and ultimately cause apoptosis indiverse cell types, including prostate cancer cells (3, 9). This proc-essing leads to cleavage of various death substrates, which in turn leadto morphological changes typical of apoptosis. There are two familiesof caspases based on the lengths of their NH2-terminal prodomains.Caspase-1, -2, -4, -5, -8, -9, and -10 have long prodomains andfunction in targeting and regulating apoptosis. Caspase-3, -6, and -7have short prodomains and are responsible for the execution ofapoptosis by operating at the downstream end of the DNA repairenzyme poly(ADP-ribose) polymerase, whose cleavage is essentialfor apoptosis induction (3, 10). Caspase activation involves a distinct,temporal cascade demonstrating a complex hierarchy within thecaspase family. Mitochondria also play a key part in the regulation ofapoptosis (11). Cytochromec, which usually is present in the mito-chondrial intermembrane space, is released into the cytosol afterinduction of apoptosis by different stimuli, including chemotherapeu-tic and DNA-damaging agents. The release of cytochromec frommitochondria and its subsequent binding to caspase-9 (resulting intransactivation of procaspase-9 by Apaf 1) can trigger the sequentialactivation of caspase-3, an apoptosis executioner (12).

Caspase 1, also known as interleukin 1b-converting enzyme, isrequired for apoptosis (7, 13). Caspase-1 is an initiator caspase thatwas originally characterized as cleaving inactive prointerleukin 1b togenerate the active proinflammatory cytokine interleukin 1b (13).Overexpression of caspase-1 has been found to induce apoptosis inmammalian and insect cells (10). Caspase-9 is another initiatorcaspase that is dependent on cytosolic factors for expression of itsactivity (14). Apoptotic signals release cytochromec from the mito-chondria where it associates with Apaf-1 in the presence of dATP.Apaf-1 recognizes the inactive procaspase-9 and forms the apopto-some, which triggers autocatalytic processing of procaspase-9 (12,15). Caspase-3 is the ultimate executioner caspase that is essential forthe nuclear changes associated with apoptosis, including chromatincondensation (4). An expanding body of recent evidence suggests thatthe caspase cascade is involved in the execution of apoptosis inprostate cancer cells in response to diverse stimuli, including lovas-tatin and Fas-mediated signaling (16, 17), TGF-b13 (18), and ocadaic

Received 8/8/00; accepted 11/20/00.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 NIH Grant R01 DK 53525-03.2 To whom requests for reprints should be addressed, at Division of Urology, Depart-

ment of Surgery, 400, MSTF, University of Maryland Medical Center, 10 South PineStreet, Baltimore, MD 21201; Phone: (410) 706-7549; Fax: (410) 706-0311; E-mail:[email protected].

3 The abbreviations used are: TGF-b, transforming growth factor-b; TUNEL, terminaldeoxynucleotidyl transferase (Tdt)-mediated nick end labeling; RT-PCR, reverse tran-scription-PCR; BPH, benign prostatic hyperplasia; GAPDH, glyceraldehyde-3-phosphatedehydrogenase.

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acid (19). In addition, blockade of caspases activity by the inhibitorCrmA has been shown to suppress androgen-ablation-induced apo-ptosis in LNCaP prostate cancer cellsin vitro and in vivo (20).Furthermore, caspase-3 activation plays a role in apoptotic inductionof other human cancers, such as osteocarcinoma (21) and ovarian(22), gastric, and breast cancer (23).

Considering the evidence gathered in this laboratory that activationof TGF-b signaling suppresses prostate tumorigenicity via inductionof caspase-1-mediated apoptosis (18, 24) and concerning the centralrole of caspase cascade in the execution of apoptosis of prostatecancer cells in response various agents (15, 16, 19), we examined thepattern of caspase-1, -3, and -9 expression in normal and malignanthuman prostate. Our findings indicate that the immunoreactivity ofcaspase-1 and -3 (but not caspase-9) is significantly decreased inprostate cancer, whereas no significant changes in mRNA expressionwere observed.

MATERIALS AND METHODS

Immunohistochemical Analysis. Formalin-fixed, paraffin-embedded tis-sue sections (6mm) from human prostate tissue were obtained from theDepartment of Pathology Archives at the University of Maryland MedicalCenter. Specimens included 42 primary prostate adenocarcinomas from pa-tients undergoing radical prostatectomy for localized disease. Prostatic tumorswere characterized for pathological grade using the Gleason scoring systemprior to immunohistochemical analysis (A. B.). Six normal prostates wereobtained from age-matched patients undergoing cystoprostatectomy for blad-der cancer.

Expression of caspase-1 was determined using rabbit polyclonal antibodyagainst human caspase-1 from Santa Cruz Biotechnology (Santa Cruz, CA).Expression of caspase-3 was determined using the antibody from PharMingen(San Diego, CA). Both antibodies recognize the precursor and the activesubunits of caspase-1 and -3, respectively. The bcl-2 mouse monoclonalantibody was obtained from DAKO (Carpinteria, CA). The proliferative indexwas determined on the basis of Ki-67 immunostaining using the mousemonoclonal MIB1 antibody (AMAC, Westbrook, ME), as described previ-ously (6, 25). Prostate tissue sections were incubated 30 min at room temper-ature with the appropriate secondary antibody (Santa Cruz Biotechnology), andcolor development was accomplished using the ABC kit (Santa Cruz Biotech-nology) and the chromogen 3,3-diaminobenzidine tetrahydrochloride solution.Negative controls were processed in the absence of primary antibody. Sectionswere counterstained with hematoxylin, and cells were reviewed at three fieldsrandomly selected at3400 by three independent observers (R. N. W., A. K.,and N. K.) who were blinded to the clinicopathological characteristics of thepatients and disease outcome. Scoring of immunoreactive cells was based onthe distribution of positive cells in three different fields (with;300 cells perfield) within the same section, and the percentage of positive immunoreactivitywas expressed as the percentage of the number of stained cells over the totalnumber of cells.

Apoptosis Detection.Detection of apoptosisin situ was performed inparaffin-embedded sections using the ApoTag Kit (Intergen, Purchase, NY),based on the TUNEL assay as described previously (25). Sections of rat ventralprostate after castration were used as biologically positive controls. Negativecontrols consisted of consecutive sections of each case in which the terminaldeoxynucleotidyl transferase enzyme was omitted. Sections were counter-stained with methyl green.

RT-PCR Analysis. RNA was isolated from cells by the TRIzol method(18). The four human prostate cancer cell lines used, PC-3, DU-145, TSU-Pr1,and LNCaP, were obtained from the American Tissue Culture Collection(Rockville, MD) and were maintained in RPMI 1640 (Life Technologies,Gaithersburg, MD) supplemented with 10% FCS (Hyclone, Logan, UT). RNAwas extracted from prostate cancer tissue as described previously (26). Thespecimens included 15 normal prostates (from patients undergoing cystopros-tatectomy for bladder cancer), 23 primary prostate adenocarcinoma specimens,8 lymph nodes negative for metastatic deposits of prostate cancer, 15 lymphnodes positive for metastatic prostate cancer (obtained from patients undergo-ing laparoscopic lymph node dissection), and 3 specimens with histological

evidence of BPH. RT-PCR was performed using 2mg of total cellular RNAand the Ribo Clone cDNA synthesis kit (Promega Corp., Madison, WI) in aPerkin-Elmer amplification cycler (Wellesley, MA). The sequence of thehuman primers used were as follows:Caspase-1: sense, 59-ATCCGTTCCATGGGTGAAGGTACA-39; an-tisense, 59-CAAATGCCTCCAGCTCTGTAATCA-39

Caspase-3: sense, 59-TTCAGAGGGGATCGTTGTAGAAGTC-39;antisense, 59-CAAGCTTGTCGGCATACTGTTTCAG-39

Caspase-9: sense, 59-ATGGACGAAGCGGATCGGCGGCTCC-39; antisense, 59-GCACCACTGGGGGTAAGGTTTTCTAG-39

The primers for humanGAPDH were obtained from Clonetech(Palo Alto, CA), and the sequences were as described previously (18).The conditions used for the RT-PCR for each caspase were as follows:for caspase-1, 94°C for 5 min, 60°C for 5 min, 40 cycles of 94°C for1 min, 60°C for 2 min, and 72°C for 2 min, with final extension of72°C for 10 min; for caspase-3, 94°C for 5 min, 35 cycles of 68°C for1 min and 94°C for 1 min, with final extension of 72°C for 10 min;for caspase-9, 94°C for 5 min, 30 cycles of 68°C for 1 min and 95°Cfor 1 min, with final extension of 72°C for 10 min. The integrity of theRNA used for RT-PCR was confirmed using GAPDH synthesis as apositive control reaction as described previously (18). The amplifiedRT-PCR products were analyzed electrophoretically through 1% aga-rose gels, visualized by ethidium bromide staining, and photographedunder UV illumination.

Western Blot Analysis. Cell lysates from the human prostate cancer celllines were lysed as described previously (18). Cell lysates were subjected toSDS-PAGE (12.5%) followed by Western blotting, using the following anti-bodies: the antibody against human caspase-1 from Santa Cruz Biotechnology;the antibody against caspase-3 from PharMingen; the antibody againstcaspase-9 from New England Biolabs (Beverly, MA); and antibody againsta-actin (Oncogene Research, Boston, MA). Protein expression was detectedusing the ECL detection kit (Amersham Int., Arlington Heights, IL). Theexpression ofa-actin was used as a normalizing control.

Statistical Analysis. Statistical analysis was conducted using thet test foranalysis of significance between the different values. Values were expressed asthe mean values6 SE. Statistical significance was established at values ofP , 0.05.

RESULTS

The expression of caspase-1, -3, and -9, three key caspases previ-ously implicated in the execution of apoptosis in prostate cancer cells(16, 18, 20), was determined in human prostate specimens by immu-nohistochemical analysis. Fig. 1 shows a characteristic caspase-1immunoreactivity pattern in normal, benign, and malignant humanprostate tissue. Uniformly intense cytoplasmic immunoreactivity forcaspase-1 was observed among epithelial cells in the normal prostate(Fig. 1). A significant decrease in caspase-1 immunostaining wasdetected in epithelial cells from a BPH prostate (Fig. 1), whereasmalignant prostatic tissue exhibited a heterogeneous pattern of dra-matically reduced caspase-1 immunoreactivity among the tumor cellpopulations (Fig. 1,C and D). A similar pattern of heterogeneousimmunoreactivity in prostate cancer tissue was observed for caspase-3(data not shown).

Quantitative analysis of the data (summarized in Table 1) revealeda dramatic loss of caspase-1 protein expression in primary prostatictumors compared with the normal gland (P , 0.01). In the prostatecancer specimens, a significant decrease in caspase-1 and -3(P , 0.01) was paralleled by a significant increase in bcl-2 levels(P , 0.05). As shown on Table 1, the proliferative index/apoptoticindex ratio was more than 2-fold higher (1.8) in the malignant prostatecompared with the normal gland (0.7), indicating the higher numberof proliferating epithelial cells in prostate cancer. Surprisingly, ouranalysis documented that the TUNEL-positive prostate cells are not

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CASPASE EXPRESSION IN PROSTATE CANCER



necessarily high caspase-1- or caspase-3-expressing cell populationsbecause linear regression revealed no correlation between a highcaspase immunoreactivity and elevated apoptotic index. Also shownon Table 1 is the significant increase in bcl-2 protein expression in theprostatic tumor epithelial cells (23.5%) compared with the normalprostate (4.5%;P , 0.05).

Table 2 shows the association of clinicopathological characteristicswith the expression levels of caspase-1, caspase-3, and bcl-2 in theprostatic tumors analyzed. A relatively high caspase-1 expression,relative to the low apoptotic index, was detected in the normal humanprostate (60%). In prostatic adenocarcinoma specimens, there was adramatic decrease in caspase-1 levels to 11% (Gleason 3–5) and 21%(Gleason 6–7; Table 2). For caspase-3 expression, there was a statis-tically significant decrease (P , 0.05) in the moderately differentiatedtumor group (Gleason 6–7; 58%) as well as the poorly differentiatedtumors (Gleason 8–9; 51%) compared with the normal human pros-tate (82%; Table 2).

Using linear regression analysis, we found no direct correlationbetween loss of caspase-1 and -3 expression and apoptotic index orproliferative index of the prostatic tumor cell populations (r 5 0.233).Although a trend was clearly detected toward higher levels ofcaspase-1 expression with increasing Gleason grade, the differencesfailed to reach statistical significance (P . 0.05).

In Fig. 2, Western analysis reveals the presence of the proenzymeforms of caspase-1, -3, and -9 in the androgen-independent PC-3,TSU-Pr1, and DU-145 and the androgen-responsive LNCaP human

prostate cancer cell line. The weak presence of the activated form ofcaspase-3 probably reflects constitutive activation in all of the celllines analyzed. Although caspase-3 and -9 proteins were detected inall four cell lines, the LNCaP and DU-145 cell lines had verylow expression of both the proenzyme and active form of caspase-1(Fig. 2).

To examine whether the loss of protein expression in prostatictumors as detected by immunohistochemistry was a result of down-regulation of mRNA expression, semiquantitative RT-PCR analysiswas performed. A total of 64 samples (15 normal prostate, 8 negativelymph nodes, 15 positive lymph nodes, 23 prostate cancer, and 3 BPHsamples) were analyzed for caspase-1, -3, and -9 expression. Thehuman prostate cancer cell lines were also analyzed. Fig. 3 shows

Table 1 Apoptosis profiling in normal and malignant human prostate

Specimens% Caspase-1expressiona

% Caspase-3expressiona

% Bcl-2expressiona

Proliferativeindex/Apoptotic

indexb,c

Normal prostate (n5 6) 59.96 8.1 82.16 3.1 4.56 1.2 0.7Prostate cancer (n5 42) 20.86 3.0d 60.76 3.2d 23.56 4.7d 1.8d

a Values represent the percentage of positive cells over the total number of cells/section. Values represent mean6 SE.

b Proliferative index is defined as the percentage of Ki-67-positive cells over the totalnumber of cells (as described in ‘Materials and Methods‘).

c Apoptotic index is defined as the percentage of TUNEL-positive cells over the totalnumber of cells.

d Values statistically significant from the normal prostate (P, 0.05).

Fig. 1. Photomicrographs of caspase-1 immunoreactivity patterns in normal (A), benign (BPH;B), and malignant (CandD) human prostate.A, abundant immunostaining is detectedin the normal prostate (magnification,350). B, a BPH section with moderate immunoreactivity for caspase-1 among the benign epithelial cells.C and D, two different prostaticadenocarcinoma sections, with characteristically extensive loss of caspase-1 immunoreactivity. The Gleason scores of the cancer foci indicated were 4 and 7 forC andD, respectively(magnification,3100).

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representative examples of the mRNA expression profile forcaspase-1, -3, and -9 in human prostatic tissue and prostate cancer celllines. The expression profile of the control mRNA GAPDH in theidentical series of samples reveals equivalent loading of the RT-PCRproducts (Fig. 3). As shown in Fig. 3A, relatively high levels ofcaspase-1 mRNA expression were detected in both the normal andmalignant prostate, as assessed by the strong intensity of the band.The expression level for caspase-3 and caspase-9 mRNA in prostatictumors was slightly lower than caspase-1. No significant differencesin the mRNA expression levels for caspase-1,- 3, or -9 were detectedbetween the normal prostate and prostate cancer specimens (Fig. 3A).

We subsequently expanded our analysis to investigate the mRNAexpression in four prostate cancer cell lines: PC-3, TSU-Pr1, DU-145,and LNCaP. As shown in Fig. 3B, caspase-3 and -9 are expressed incomparable levels in all four cell lines analyzed. Interestingly how-ever, the mRNA expression levels for caspase-1 were undetectable inthe androgen-independent DU-145 and the androgen-sensitive LNCaPprostate cancer cells (Fig. 3B). This observation was in accord withthe lack of caspase-1 protein expression in these two cell lines asdetected by Western blotting (Fig. 2).

DISCUSSION

Activation of the caspase cascade has been correlated with the onsetof apoptosis, and caspase inhibition attenuates apoptosis in prostatecancer cells in response to diverse apoptotic stimuli, including andro-gen ablation (8, 16, 17, 19, 20). In this study, we demonstrate adiminished detection of caspase-1 and -3 protein in human prostatecancer compared with the normal gland with no significant changes in

the mRNA expression. Caspase-1 and -3 immunoreactivity was pre-dominantly localized to the secretory epithelial cells of the prostate.This is similar to an earlier report indicating expression of caspase-3to the prostatic epithelium of the normal gland (27). Our data indicatea high degree of variation in the immunostaining and pattern ofexpression of caspase-1 and -3 in prostatic tumor epithelial cells thatwas not correlated with the incidence of apoptosisin situ. Moreover,there was no statistically significant correlation between eithercaspase-1 or caspase-3 expression with Gleason grade of the prostaticadenocarcinomas examined. The possibility that analysis of a largergroup of tumors with more cases in the low and high histologicalgrade categories may yield differences that achieve statistical signif-icance cannot be ruled out. Conceptually, the present findings are incontrast with a recent report indicating a correlation betweencaspase-3 expression and elevated apoptosis and histological aggres-sion in breast cancer (28). On the other hand, the high proliferativecapacity and reduced apoptosis, which correlated with the elevatedbcl-2 expression of the epithelial cells in prostatic tumors, are in fullaccord with previous studies on the kinetics of prostate cancer growth(5, 25, 29).

The relatively high caspase-1 and -3 immunoreactivity detected inthe human prostate tissue compared with the low apoptotic indices ofthe same tumor cell populations probably reflects the recognition ofboth the active and inactive (constitutively expressed) forms of theenzyme by the antibody in paraffin-embedded sections. This possi-bility is somewhat challenged by the results of the Western analysis inprostate cancer cells not actively undergoing apoptosis, which re-vealed endogenous expression of only the proenzyme form ofcaspase-1, whereas the active caspase-1 was not detected. Cell lysatesfrom prostatic tumors are currently being analyzed to investigate theexpression of the active as well as the inactive forms of the caspases(caspase-1, -3, and -9) in clinical prostate cancer specimens frompatients treated with hormonal ablation and radiotherapy.

The present data indicating no significant changes at the mRNAlevel, although based on semiquantitative analysis, are consistent withthe possibility that changes in the levels of inactive caspases, ratherthan down-regulation of the active enzymes are responsible for thereduced caspase-1 immunoreactivity detected. Although this evidencemay point to a potential posttranscriptional deregulation of caspaseexpression, RNase protection assays will provide a better insight intothe level of control. Furthermore, our observations indicating that theandrogen-responsive LNCaP and the androgen-independent DU-145prostate cancer cells both lack expression of caspase-1 at the mRNAand protein levels highlight the functional significance of caspase-1 inthe development of prostate tumorigenic phenotype. In accord withthis concept is existing evidence that LNCaP cells are resistant toradiation-induced apoptosis (30). The effect of genetic restoration ofcaspase-1 expression on the tumorigenic potential and apoptotic “sen-sitivity” of these cells is currently being investigated.

The active role of caspase-1 (originally thought to be involved onlyin the proinflammatory response) in the apoptotic program is evolvingrapidly. Although studies with caspase-1-deficient mice indicated no

Table 2 Association of caspase-1, caspase-3, and bcl-2 protein expressiona with the apoptotic status and tumor grade of prostate cancer

Specimensb % Caspase-1 expression % Caspase-3 expression Proliferative index (%)Apoptoticindex (%) % Bcl-2 expression

Normal prostate (n5 6) 59.96 8.1 82.16 3.1 2.46 0.5 3.66 1.1 4.56 1.2Prostate cancer

Gleason 3–5 (n5 6) 11.56 4.0c 79.96 7.9 1.76 0.8 1.86 0.9 23.56 4.7c

Gleason 6–7 (n5 31) 21.66 3.7c 58.76 3.5c 3.66 0.3c 5.26 0.7 38.76 3.3c

Gleason 8–9 (n5 5) 26.66 5.8c 51.66 5.4c 10.46 1.3c 7.56 2.2c 26.96 7.6c

a Values represent the percentage of positive cells over total number of cells/section (mean6 SE).b n, number of specimens analyzed per group.c Statistically significant difference from values for the normal prostate (P, 0.05).

Fig. 2. Western blot analysis of caspase protein expression in human prostate cancercell lines. Cell lysates were prepared from the various prostate cancer cell lines andsubjected to PAGE (12.5%) as described in “Materials and Methods.”Lane 1, low-rangemolecular weight marker;Lane 2, blank;Lanes 3–6, cell lysates from PC-3, LNCaP,TSU-Pr1, and DU-145 cells, respectively. The molecular weights of caspase-1,-3, and -9proenzymes are indicated on theleft. Expression ofa-actin was used as a normalizingcontrol to confirm equivalent protein loading and transfer.

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differences in their apoptotic response to Fas/CD95 signaling (31),evidence from this laboratory indicated that TGF-b1-mediated apo-ptosis induction in prostate cancer cells proceeds via caspase-1 acti-vation (18). The concept was further supported by a recent studydemonstrating the functional significance of caspase-1 in enhancingFas-mediated apoptosis potentially through facilitation of caspase-8

activation (32). The mechanism of potential synergy betweencaspase-1 and cytochromec is unknown at present, but it may wellrequire active caspase-8 to amplify a caspase-9 cascade. Becauseprostatic tumors overexpress bcl-2 oncoprotein (Refs. 25, 29, andpresent data), which restricts cytochromec release and consequentactivation of caspase-9, thus incapacitating apoptosis activation inprostate cancer, identification of the effect of high bcl-2 levels oncaspase-9 expression/activation pattern in malignant prostate epithe-lial cells might be worth pursuing.

The present study provides the first evidence to indicate a signifi-cant loss of caspase-1 and -3 protein expression in human prostatictumors compared with the normal gland and to implicate deregulatedexpression of caspases in prostate tumorigenesis. Indirect support forthis concept stems from a report that documented deregulation ofinterleukin 1b-converting enzyme-like caspase expression in prostatecancer patients treated with antiandrogens (33). In accord with ourresults in prostate cancer, recent studies by Jarryet al. (34) demon-strated significant down-regulation of caspase-1 expression in humancolon cancer. In contrast, an up-regulation of caspase-1 protein hasbeen reported in pancreatic tumors (35). The apparent discrepancymay reflect specific differences in the molecular mechanisms under-lying the development of various human malignancies, or alterna-tively may be attributable to the different origin of the antibody usedin the latter study. The diversity of mechanisms involved in theregulation and implementation of apoptosis, which is indicative of arich variety of targets for its inhibition during tumorigenic develop-ment, must also be considered (36).

Growing evidence implicates deregulation of caspase-1 and -3 inthe pathophysiology of other human diseases. A potentially importantrole for caspase-1 has been suggested in the pathogenesis of Hunting-ton’s disease (37), whereas caspase-3 has been functionally implicatedin the ultimate death of neurons by apoptosis in Alzheimer’s disease(38). Mice deficient in caspase-3 exhibit specific defects in the apo-ptotic pathway, including delayed kinetics, and lack of DNA frag-mentation during brain development (4). Furthermore, caspase-2 and-3 are expressed in Hodgkin’s and non-Hodgkin’s lymphomas,chronic lymphocytic leukemias, and reactive lymph nodes (39, 40),and more recent evidence supports a predictive role for caspase-3localization in the clinical outcome of B-cell lymphoma (41).

In conclusion, our observations provide a rationale for the involve-ment of a deregulated caspase cascade in prostate tumorigenesis. Asmight be predicted, loss of expression of key caspases would conferprotection against apoptosis in malignant prostate cells. The presentfindings may have high clinical relevance by identifying a potentiallysignificant value for caspases not only as markers of disease progres-sion, but also as therapeutic targets for effective activation of theapoptotic process in advanced prostate cancer. Prospective studiesinvolving a large number of clinical tissue specimens from patientswith advanced disease before and after hormonal therapy are requiredto establish the potential use of caspases in the development oftherapeutic modalities for advanced prostate cancer.

ACKNOWLEDGMENTS

We thank Dr. Stephen C. Jacobs (Division of Urology) for providing freshprostate tumor tissue for RNA analysis, Dr. Yanping Guo for useful advice withthe caspase-1 immunostaining, and Jordan Lerner, Medical Media, Baltimore VAMedical Center, for help with the preparation of the color illustrations.

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Fig. 3.A, RT-PCR analysis of mRNA expression of caspase-1, -3, and -9 in normal andmalignant human prostate.Lane 1, thewX174 molecular weight marker;Lane 2, blank;Lanes 4and5, negative and positive (for metastatic deposits) lymph nodes, respectively;Lanes 6–9, individual normal human prostate specimens;Lanes 3and 10–13, primaryprostate adenocarcinoma specimens. GAPDH mRNA expression was serially analyzed forall of the samples as a normalizing control. The sizes of individual bands correspondingto the specific mRNA species are shown in theright. B, expression profile of mRNA forcaspases-1, -3, and -9 in human prostate cancer cell lines. RT-PCR analysis was per-formed using the specific primers for each caspase as described in “Materials andMethods.”Lane 1, thewX174 molecular weight marker;Lane 2, blank;Lanes 3–6, PC-3,TSU-Pr1, DU-145, and LNCaP cells, respectively;Lane 7, human prostate adenocarci-noma specimen. GAPDH was used to normalize for mRNA integrity and equivalentloading.

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2001;61:1227-1232. Cancer Res Rachel N. Winter, Andrew Kramer, Andrew Borkowski, et al. Human Prostate CancerLoss of Caspase-1 and Caspase-3 Protein Expression in

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