[CANCER RESEARCH 58, 2703-2706, July I, 1998]

Advances in Brief

Interleukin 1/3-converting Enzyme (Caspase-1) Is Overexpressed inAdenocarcinoma of the Pancreas1

Susanne Gansauge,2 Frank Gansauge,2 Yinmo Yang, JörgMüller,Thomas Seufferlein, Marco Ramadan!, andHans G. Beger3

Division of Molecular Oncology, Department of General Surgery, University of Vim, 89075 Ulm, Germany ¡S.G., F. G., Y. Y., J. M., M. R., H. G. BJ: Department ofGastroenterology, University of Ulm, 89081 Ulm, Germany ¡T.S.¡;and Department of Surgery, Beijing Medical University, Beijing 100034, People's Republic of China ¡Y.Y.]

Abstract

We investigated the expression of interleukin Iß-converting enzyme(ICE,- caspase-1) in human adenocarcinomas of the pancreas. Inumino-

histochemistry and Western blot analyses revealed an overexpression ofICE in 71 and 80% of tumor cells, respectively. Also, on a mRNA level,ICE mRNA was overexpressed in 45% of the cases, as compared tonormal pancreatic tissue. Interestingly, the overexpression of ICE intumor cells correlated significantly with the overexpression of cyclin Dl,epidermal growth factor, and epidermal growth factor receptor(P < 0.0005, P < 0.05, and P < 0.002, respectively), which are involved incell cycle progression and proliferation in human pancreatic carcinoma.This is the first report concerning ICE expression in human carcinomas;however, the exact mechanism underlying these close correlations warrantfurther research.

Introduction

ICE,4 now named caspase-1, is a cysteine protease that cleaves

inactive pro-IL-lß to generate the active Mr 17,500 proinflammatorycytokine IL-Iß(1). ICE was first isolated from the human monocytic

cell line THP 1 as an enzyme composed of Air 10,000 (plO) and Mr20,000 (p20) subunits. It is expressed in many tissues as an inactiveMr 45,000 precursor protein (p45), from which the active enzymesubunits are derived by an autocatalytic cleavage process (2).

Sequence homology between ICE and the death gene product CED-3

of the nematode Caenorhabditis elegans suggests that mammalian ICEmight also be involved in apoptosis (3). Overexpression of ICE in ratfibroblasts leads to programmed cell death (4), and coexpression of thecrmA protein, a potent ICE inhibitor, blocks ICE-induced apoptosis (5).Similarly, the BCL-2 protein, which is known to have a suppressiveinfluence on apoptotic processes, inhibits ICE-dependent apoptosis (6).

Furthermore, ICE is involved in neuronal cell death induced by nervegrowth factor depletion, as well as in Fas-mediated apoptosis (5, 7).Additionally, ICE is expressed in cisplatin or IFN-y-induced apoptosis (8,

9). However, ICE is not the only protease in the normal apoptoticpathway because thymocytes and macrophages from ICE-deficient mice

undergo apoptosis normally (10).Recently, several new members of the family of ICE-like proteases

have been identified and characterized, and overexpression of any ofthe ICE-family proteases results in increased levels of apoptosis in avariety of cell types (11-14). To assess the role of the "mammalian

Received 3/19/98: accepted 5/15/98.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 with18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by Deutsche Krebshilfe Grant 10-1276-Gal (to S. G. and

F. G.).2 The first two authors contributed equally to this paper.1To whom requests for reprints should be addressed, at Department of General

Surgery. University of Ulm, Steinhövelstrasse 9, 89075 Ulm, Germany. Phone:49 731 502 7200; Fax: 49 731 502 7209.

4 The abbreviations used are: ICE, interleukin Iß-convertingenzyme; IL-lß,interleu

kin 1/3; EOF, epidermal growth factor; EGF-R, EOF receptor.

death gene" ICE in pancreatic cancer, we investigated the ICE ex

pression on protein and mRNA levels in tissues of pancreatic adeno-

carcinoma. Interestingly, we found an overexpression of ICE in human pancreatic cancer and very close correlations between ICE,cyclin D, EOF, and EGF-R expression in pancreatic tumor cells,

pointing to possible aspects of ICE in proliferative processes in humanpancreatic carcinoma cells.

Materials and Methods

Tissue Samples. Pancreatic carcinoma tissues were obtained from 42 patients who underwent surgical operations for pancreatic cancer at the Department of General Surgery, University of Ulm. The group of patients included 20female and 22 male patients. The median age was 61.8 years (range, 38-78years). Tissues were collected after surgical removal, immediately snap-frozenin liquid nitrogen, and stored at -80°C, or they were fixed in 4% formalin for

1 day at room temperature, processed, and embedded in paraffin. Forty-two

adenocarcinoma tissues, 3 normal tissues from organ donors, and 4 normalpancreatic tissues from patients undergoing surgery for pancreatic cancer wereused for immunohistochemistry. and with 20 pancreatic carcinoma tissues and7 normal pancreas tissues, we performed reverse transcriptase-PCR and West

ern blot analysis.Immunohistochemistry. Five-jim paraffin sections were adhered to si-

lanized slides, deparaffinized, and hydrated by passage through xylene (threetimes for 5 min each), a graded series of ethanol (one time each at 100, 80, 70,and 50%) for 5 min, and distilled water for 10 min. Endogenous peroxidaseactivity was blocked with 3% hydrogen peroxide in methanol (30 min) between the first two steps of washing with ethanol. After each following step,sections were washed with 0.5 M Tris buffer (pH 7.6), three times for 10 min.The tissue sections were covered with 5% normal goat serum (DAKO,Glostrup. Denmark) in Tris-buffered saline for 60 min and were incubated

overnight with the polyclonal rabbit antihuman ICE antibody (Upstate Biotechnology, Lake Placid, NY) in a dilution of 1:100. For each case, a corresponding section was incubated in Tris-buffered saline without the primary

antibody as a control for nonspecific staining. Further negative controls consisted of normal rabbit serum instead of specific antisera. Biotinylated pigantirabbit secondary antibody was added for 45 min followed by the avidin-

biotinylated peroxidase complex for an additional 45 min. After washing withdistilled water for 10 min, staining was achieved by using 3,3'-diaminoben-zidine. The sections were then counterstained with Mayer's hematoxylin and

mounted. Staining of cyclin Dl, EOF, and EGF-R was achieved by using themonoclonal antibodies DCS-6 (anti-cyclin DI; Oncogene Science. Cambridge,MA), anti-EGF-R clone-528 (Oncogene Science), and the polyclonal rabbitserum Ab-3 (anti-EGF; Oncogene Science), as described elsewhere (15, 16).For staining of caspase-2 and caspase-3, which was performed in 12 carcinomatissues and 3 tissues from normal pancreas, we used the antibodies ICH-1S/L(H-19, polyclonal rabbit antihuman caspase-2; Santa Cruz Biotechnology,Santa Cruz, CA) and CPP 32 (N-19, polyclonal goat antihuman caspase-3;

Santa Cruz Biotechnology).Reverse Transcription and PCR. Frozen pancreatic tissue was cut in

small blocks, which were examined by hematoxylin staining on the amount oftumor cells and tissue of chronic pancreatitis. From 100 mg of 20 tissues,which showed >30% tumor cells and < 10% chronic pancreatitis, we isolatedmRNA using a guanidinium thiocyanate method and oligo(dT)-cellulose col

umn chromatography (QuickPrep Micro mRNA Purification Kit; Pharmacia

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ICE IN PANCREATIC CANCER

Biotech). The mRNA was dissolved in the elution buffer provided in the kit ina final volume of 30 ^tl. cDNA was prepared by reverse transcription of mRNAusing Superscript RT RNase H-Reverse Transcriptase (Life Technologies,Inc.) and diluted with distilled water to a final volume of 40 /j.1.A 399-bp ICEfragment was amplified using following primers: 5'-GGAAATTACCTTA-ATATGCAAGAC-3' (sense) and 5'-CATGAACACCAGGAACGTGCT-

GTC-3' (antisense). A 5-ju.l aliquot of cDNA was amplified under followingcycling conditions: 94°Cfor 60 s, 55°Cfor 30 s, and 72°Cfor 120 s for 35

cycles. The PCR products were electrophoresed through a 1% agarose gelcontaining ethidium bromide and visualized by UV rays. /3-Actin was used as

an internal standard to confirm equal loading in each experiment.Western Blot Analysis. Pancreatic tissues were lysed and dissociated by

sonication. Then the lysates were boiled in SDS-PAGE sample buffer for 5

min. Thirty ;ug of protein were electrophoretically resolved on denaturating15% polyacrylamide gels with a 3% stacking gel. Proteins were transferred tonitrocellulose membranes (Schleicher & Schiill, Dassel, Germany) using atransblot apparatus (Phase, Lübeck,Germany). Nonspecific interactions wereblocked by preincubation of the membranes with a milk powder suspension(10% dry milk in PBS) overnight at 4°C.After incubation of the membranes

with monoclonal antibodies the binding of antibodies was detected using theECL system (Amersham). The monoclonal antibody CAL directed againsthuman ICE (caspase-1) was a generous gift from Dr. C. Miossec (Hoechst

Marion Roussel, Romainville, France). The autocleavage experiments of themonocytic cell line THP 1 was performed as described recently (17).

Results

ICE Is Expressed in Pancreatic Carcinoma Tissue but not inNormal Pancreatic Tissue. The immunohistochemical staining ofhuman pancreatic tissues revealed a clear overexpression of ICE in

tumor cells as compared to normal pancreatic tissues, where noICE-immunoreactivity was observed (Fig. 1/4). Using a polyclonal

rabbit antiserum recognizing human ICE, a moderate and predominantly cytoplasmatic ICE expression was detected in 71% of thepancreatic tumors (Fig. Iß).The results of the stainings were confirmed by staining of cryostat sections of the same tumors using themonoclonal antibody CAL, which was kindly provided by Dr. C.Miossec. In chronic pancreatitis tissue, which often surrounds thepancreatic carcinoma (55%), we could demonstrate that, in 96%, ICEexpression was different from that in tumor cells in that the stainingwas stronger than in carcinoma cells, especially in cells of ductalorigin, where a positive cytoplasmatic and nuclear ICE expressioncould be observed (Fig. 1C). Dying cells showing a pyknotic nucleusalways expressed the ICE protein (Fig. ID).

To assess the specificity of the ICE expression, we also performedimmunohistochemical stainings using antibodies directed against twoother ICE-like proteases (caspase-2 and caspase-3) in 12 of the

pancreatic carcinoma tissues and 3 of the normal pancreatic tissues. Innormal pancreas, a faint staining of ductal cells was observed usingthe polyclonal antibody directed against caspase-2 and caspase-3. Inpancreatic carcinoma tissues, caspase-2 and caspase-3 were both

overexpressed in 8 of 12 (67%) tissue samples. Interestingly, thestaining patterns of ICE (caspase-1), caspase-2, and caspase-3 were

nearly identical in the tissues investigated (data not shown).Western Blot Analysis of ICE in Pancreatic Cancer. To confirm

the overexpression of ICE seen in immunohistochemical stainings, weperformed Western blots with the monoclonal antibody CAL against

Fig. 1. Expression of ICI: in human pancreatic carcinoma and in normal pancreatic tissue. A. in normal pancreatic tissue, no staining was achieved in immunohistochemislry usingthe polyclcmal rahhil antihuman ICH antiscrum. whereas in 71% of human pancreatic carcinoma tissues (0). staining of pancreatic carcinoma cells was achieved. C, in 96% of caseswith obstructive chronic pancreatitis, which surrounded pancreatic cancer in 55*, a strong ICE expression was found mainly in cells of ductal origin. Magnification, X40 (A-Q. D,in dying cells with pyknotic nuclei, ICH expression was always (bund. Magnification, X100.

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ICE IN PANCREATIC CANCER

kDa

45 —¿�

30

i 8 10 11

Fig. 2. Western blot analysis of ICE in pancreatic cancer and normal pancreatic tissue.In normal pancreatic tissue (Lanes 3 and 4), no signal was obtained. Lysates from themonocylic cell line THP 1 served as positive control (Lane I ). To determine autocleavageof ICE, the THP 1 lysate was incubated at room temperature for 4 h (Lane 2). In 80% oflysates from pancreatic cancer (Lanes 5-11), a M, 45,000 band was detected by themonoclonal anti-ICE antibody CAL. Furthermore, in 60% of the pancreatic cancer lysates.

an additional band was detected at M, 30,000.

human ICE with pancreatic tumor tissues. In 80% of these probes,CAL detected a band migrating at Mr 45,000 (Fig. 2), suggesting thatthere was a A/r 45,000 precursor protein of ICE. The antibody CALwas generated to detect the Mr 20,000 active form of ICE (17). Aspositive control, we used lysates from the THP 1 cell line. In THP 1lysates, a band migrating at Mr 45,000 was detected. Furthermore, aband migrating at Mr —¿�35,000-40,000 was observed. After incuba

tion of the lysate for 4 h at room temperature, the signal at Mr 45,000was absent, whereas the signal between Mr 35,000 and Mr 40,000 wasenhanced, suggesting ICE proteolysis as described recently (17).However, after 24 h of incubation at room temperature, a very faintband at M, 20,000 was observed (data not shown). In pancreaticcarcinoma tissue, this band was not detectable, but 60% of the probesshowed a signal at A/r 30,000 that was absent in the THP 1 cells. Inpancreatic tissues of healthy organ donors, no signals were obtainedusing CAL, suggesting an overexpression of the ICE protein inpancreatic adenocarcinoma tissues.

Overexpression of ICE mRNA in Human Pancreatic Carcinoma. The expression of ICE mRNA was determined by reversetranscriptase-PCR amplifying a 399-bp product (Fig. 3). In compari

son to healthy pancreatic tissues, ICE mRNA was overexpressed in45% of the samples. Immunohistochemistry revealed positivity in atleast one tissue entity in almost every sample, and Western blotanalysis yielded specific protein bands in 80% of the samples, pointing to a possible translational stabilization of the ICE protein. However, in normal pancreatic tissue no staining was achieved usinganti-ICE antibodies, and in PCR analysis, only a weak ICE mRNA

signal was obtained.Expression of ICE in Pancreatic Carcinoma Cells Correlates

Significantly with Cyclin Dl, EGF, and EGF-R Expression. Re

cently, we described the expression of cyclin Dl in human pancreaticcarcinoma (15). Interestingly, comparison of ICE and cyclin Dlexpression in the tumor cells revealed a highly significant correlationbetween the expression of these two molecules in tumor cells in ourseries of 42 human pancreatic carcinoma tissues (P < 0.0005). However, in cells with pyknotic nuclei, the monoclonal antibody DCS6against cyclin Dl protein never stained positive. Similar to the correlation to cyclin Dl, we found a close correlation between ICEexpression and the expression of EGF and EGF-receptor in pancreatic

carcinoma cells (P < 0.05 and P < 0.002, respectively), whereas nocorrelation was found between ICE and cERB-B2 expression(P = 0.29, not significant).

Correlation of ICE Antigen Expression in Tumor Cells withClinicopathological Features. With regard to clinical data, no correlation was found between the expression of ICE in tumor cells andsex, age, tumor stage, tumor size, or tumor grading. Also, no statisticalsignificance was found with regard to survival. The group of patientswhose tumors stained negatively for ICE had a median survival time

of 14.8 months, and patients with ICE-positive pancreatic adenocar-cinomas had a median survival time of 12 months (Kaplan-Meier

regression analysis, not significant).

Discussion

The ICE family of proteases plays an important role in apoptoticprocesses; however, the physiological roles of the different homologues during apoptosis are still unclear. We assessed the expressionof ICE in pancreatic carcinoma and correlated it with the expressionof (a) cyclin Dl, which is known to be a G, protein involved in cellproliferation, and (b) EGF and EGF-R, which are suggested to play a

crucial role in autocrine stimulation of human pancreatic carcinoma(18). To our knowledge, this is the first report elucidating the expression of this molecule in carcinoma cells in cancer tissues. In immu-

nohistochemistry and Western blot analyses, we never achieved ICEpositivity in normal pancreatic tissues, whereas in pancreatic carcinoma tissues, immunohistochemistry revealed positive ICE stainingof pancreatic tumor cells in 71% of the tissues. Chronic pancreatitistissue surrounding the pancreatic tumor cells expressed ICE in almostevery sample. ICE is described as a cytosolic protein, but in ourexperiments, we also could show a nuclear staining of cells in chronicpancreatitis tissue. However, most of the proven substrates for ICE-

like enzymes in apoptosis are structural or catalytical nuclear proteins,the cleavage fragments of which are found in apoptotic bodies (19),suggesting that cleavage is a nuclear event. Also, dying cells withpyknotic nuclei always stained positive with the antibody against theICE antigen. Most of the regions of chronic pancreatitis in the pancreatic tumor tissue showed strong fibrosis, implicating massive celldeath of the pancreatic cells. In these parts of the pancreatic cancertissues, as well as in the cells with pyknotic nuclei, the ICE expressionmay be indicative for ongoing apoptotic processes. In tumor cells,however, explanation of cytoplasmatic ICE expression in associationwith apoptosis seems to be more problematic because it could not beassumed that, in some tumors, nearly all cancer cells show apoptoticfeatures. Interestingly, the expression of ICE in pancreatic tumor cellscorrelated significantly with the expression of the cyclin Dl, EGF,and EGF-R antigens.

It is known that some growth factors, including EGF, stimulatecyclin Dl (20), and recently, it has been shown in a transgenic mousemodel that overexpression of cyclin Dl was associated with EGF-Ralterations (21). Besides the correlation of EGF. EGF-R, and cyclin

ICE

399 bp

ß-Actin

289 bp

123456 789 10Fig. 3. Expression of ICE mRNA in human pancreatic carcinoma and normal pancreas.

mRNA from THP 1 were used as positive control (Lane 10). showing a 399-bp product.In comparison to normal pancreatic tissues (Lanes 7-9), in pancreatic carcinomas (Lanes1-6), an overexpression was observed in 45% (as shown in Ltmes 1-4), whereas in 55%

of the pancreatic carcinomas, the faint signal obtained (Lanes 5 and 6) was not differentfrom normal pancreatic tissues. Equal loading was confirmed by ß-actinmRNA analysis.

123456 789 10

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ICE IN PANCREATIC CANCER

D l, there are also reports about the correlation of ICE expression withEGF-induced apoptosis (22). In some cells, like A431 and MDA-MB-

468 cells, EOF is able to inhibit cell growth and to induce apoptosis(23). Chin and coworkers (22) recently demonstrated that activation ofPTK-signal transducers and activators of transcription and cell cycleinhibitor p2iWAF/CIPI expression through activation of signal trans

ducers and activators of transcription may be responsible for theinduced growth arrest of EOF (24) and that ICE expression is necessary for this process. However, stimulation of the pancreatic carcinoma cell lines AsPC-1 and BxPC-3 with supramaximal concentrations of EGF or TGF-a (both 50 ng/ml) induced cell proliferation

starting after 18 h, without inducing apoptosis during an observationperiod of 48 h (data not shown).

With regard to the fact that cyclin Dl, EGF, and EGF-R overex-

pression correlates with poor prognosis in human pancreatic carcinoma (15, 25), it is hard to explain that these factors should reflect theapoptotic potential of the tumors. Also, the close correlation betweenp21wAFi/cipi md çycïmDl that we have demonstrated recently (15)could be due to the need for p2iWAF1/CIPI for stabilization of the

interactions between cyclin-dependent kinase 4 and cyclin Dl pro

moting the formation of active kinase complexes (26).The overexpression of ICE demonstrated in immunohistochemistry

was confirmed by Western blotting. We detected specific bands at Afr45,000 and Mr 30,000, suggesting that the Mr 45,000 signal representsthe immature precursor p45. The M, 30,000 signal could represent thep30 precursor lacking the 119 NH2-terminal amino acids (2, 27).

These results agree with former publications showing that the ICEprecursors are the predominant form expressed in peripheral bloodmononuclear cells and in hematopoietic cell lines (17). Even duringactive IL-lß secretion by these cells, neither the p20 nor the plO

subunit of the active ICE complex could be detected (17), suggestingthat very limited amounts of mature ICE protein are sufficient for atleast some activities ~of ICE. Because caspase-2 and, especially,caspase-3, which is known to be expressed in Hodgkin's lymphomas,non-Hodgkin's lymphomas, chronic lymphocytic leukemias, and re

active lymph nodes (28, 29), were also expressed in the majority ofhuman pancreatic carcinomas, this could point to a general activationof caspases in human pancreatic carcinoma.

In conclusion, we have shown that ICE is overexpressed in pancreatic carcinoma tissue and that the overexpression of ICE in tumorcells correlates significantly with cyclin Dl, EGF, and EGF-R. Nev

ertheless, the exact mechanisms underlying these close correlationswarrant further research.

Acknowledgments

We thank Erika Schmid for expert technical assistance and Dr. C. Miossec(Hoechst Marion Roussel, Romainville, France) for providing us with themonoclonal antibody CAL directed against human ICE (caspase-1).

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1998;58:2703-2706. Cancer Res   Susanne Gansauge, Frank Gansauge, Yinmo Yang, et al.   in Adenocarcinoma of the Pancreas

-converting Enzyme (Caspase-1) Is OverexpressedβInterleukin 1

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