Interferon ª inhibits growth of human pancreatic carcinoma ...pressor interferon regulatory factor 1 (IRF-1). IFN-ª treatment profoundly in-hibited anchorage dependent and inde-pendent

Interferon ã inhibits growth of human pancreaticcarcinoma cells via caspase-1 dependent inductionof apoptosis

K M Detjen, K Farwig, M Welzel, B Wiedenmann, S Rosewicz

AbstractBackground and aims—The poor progno-sis of pancreatic cancer is partly due toresistance to a broad spectrum of apop-totic stimuli. To identify intact proapop-totic pathways of potential clinicalrelevance, we characterised the eVects ofinterferon ã (IFN-ã) on growth and sur-vival in human pancreatic cancer cells.Methods—IFN-ã receptor expression andsignal transduction were examined byreverse transcriptase-polymerase chainreaction (RT-PCR), immunoprecipita-tion, western blot analysis, and transacti-vation assays. EVects on cell growth andsurvival were evaluated in terms of cellnumbers, colony formation, cell cycleanalysis, DNA fragmentation, and poly-(ADP ribose) polymerase (PARP) cleav-age.Results—All four pancreatic cancer celllines examined expressed functionalIFN-ã receptors and downstream eVec-tors, including the putative tumour sup-pressor interferon regulatory factor 1(IRF-1). IFN-ã treatment profoundly in-hibited anchorage dependent and inde-pendent growth of pancreatic cancer cells.Cell cycle analyses revealed subdiploidcells suggesting apoptosis, which was con-firmed by demonstration of DNA frag-mentation and PARP cleavage. Time anddose dependency of apoptosis inductionand growth inhibition correlated closely,identifying apoptosis as the main, if notexclusive, mechanism responsible forgrowth inhibition. Apoptosis was pre-ceded by upregulation of procaspase-1and accompanied by proteolytic activa-tion. Furthermore, the caspase inhibitorz-vad-fmk completely prevented IFN-ãmediated apoptosis.Conclusions—These results identify anintact proapoptotic pathway in pancreaticcancer cells and suggest that IRF-1 and/orprocaspase-1 may represent potentialtherapeutic targets to be further explored.(Gut 2001;49:251–262)

Keywords: interferon ã; apoptosis; caspase-1;interferonregulatory factor; pancreatic cancer

The type II cytokine interferon ã (IFN-ã) iscapable of eliciting potent growth inhibitoryeVects in a number of tumour models in vitroand in vivo.1–5 Direct actions of IFN-ã ontumour cells as well as indirect mechanisms

such as immunomodulation6 and antiangio-genesis7 contribute to antiproliferative actions.However, direct eVects appear to be highly tis-sue and cell type specific. As IFN-ã receptorsare relatively ubiquitously expressed in diVer-ent cell types,8 specific loss of biologicalresponsiveness in the context of malignanttransformation has been suggested and hassubsequently been demonstrated in lungtumour, melanoma, and endothelial cells.9–11

IFN-ã exerts its biological eVects via crosslinking of a heterodimeric receptor, therebycausing tyrosine phosphorylation of the recep-tor associated tyrosine kinases Jak-1 and Jak-2.These in turn phosphorylate the latent cy-tosolic transcription factor Stat-1 at tyrosineresidues (reviewed by Bach and colleagues8 andPlatanias and Fish12). On phosphorylation,Stat-1 proteins convert from the latent to theDNA binding state, dimerise, and translocateto the nucleus where they activate transcriptionfrom IFN-ã response elements (GAS) and thusinduce changes in gene expression that arethought to account for the pleiotropic cellulareVects of IFN-ã.13

Key regulators of cellular growth controlhave been shown to be diVerentially regulatedby IFN-ã in diverse cell models, leading to dis-parate biological responses such as G1arrest,14–17 S phase retardation,14 18 or inductionof apoptosis.19 In some models, the anti-mitogenic actions of IFN-ã have been furtherdissected at the cell cycle level and cell cyclearrest in the early or late G1 phase was linkedto accumulation of the hypophosphorylatedgrowth restrictive form of the tumour suppres-sor protein retinoblastoma protein (Rb). Thisfunctional activation of Rb was attributed todownregulation of G1 cyclins18 and/or induc-tion of cyclin dependent kinase inhibitors(CKIs) such as p21cip116 20 21 and/or p27kip1.22 23 Inaddition, induction of the double strandedRNA activated protein kinase (p68 PKR)24 anddownregulation of cellular c-myc25 levels werereported, although these events were not asso-ciated with phase specific cell cycle changes.

Abbreviations used in this paper: RT-PCR, reversetranscriptase-polymerase chain reaction; PARP,poly(ADP ribose) polymerase; TNF-á, tumournecrosis factor á; DTT, dithiothreitol; IRF, interferonregulatory factor; Rb, retinoblastoma protein; CKI,cyclin dependent kinase inhibitor; PBS, phosphatebuVered saline; PBST, phosphate buVeredsaline+0.1% Tween; FCS, fetal calf serum; PVDF,polyvinyl difluoride; SDS-PAGE, sodium dodecylsulphate-polyacrylamide gel electrophoresis.

Gut 2001;49:251–262 251

Medizinische Klinikmit SchwerpunktHepatologie undGastroenterologie,UniversitätsklinikumCharité, CampusVirchow Klinikum,Humboldt Universitätzu Berlin,Augustenburger Platz1, 13353 Berlin,GermanyK M Detjen†K Farwig†M WelzelB WiedenmannS Rosewicz

Institut fürPharmazie, FreieUniversität Berlin,Berlin, GermanyK Farwig

†These authorscontributed equally tothis work.

Correspondence to:Professor S [email protected]

Accepted for publication29 January 2001

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Recent interest has focused on IFN-ã medi-ated induction of apoptosis. Apoptosis or pro-grammed cell death is an active tightlyregulated process characterised by a set ofmorphological and biochemical changes, in-cluding chromatin condensation, internucleo-somal DNA cleavage, membrane blebbing, andthe formation of apoptotic bodies.26 27 In spiteof diverse stimuli, apoptotic pathways eventu-ally converge in the activation of a cascade ofwell conserved cysteine proteases (caspases)which act as principle executioners of theapoptotic process.28 29 Caspases in general areexpressed as inactive proenzymes and becomeproteolytically activated in response to proap-optotic stimuli. Under physiological condi-tions, apoptotic cell death is induced predomi-nantly by stimulation of death domaincontaining receptors such as Fas (CD 95) ortumour necrosis factor á (TNF-á) whichhave been demonstrated to be regulated byIFN-ã.30 31 Also, modulation of HLA expres-sion by IFN-ã indirectly regulates apoptosis bytargeting immunosurveillance mechanisms.32

In contrast, the relevance of direct proapototicactions of IFN-ã in tumour cells remains con-troversial, based on the striking diversity ofbiological eVects in diVerent tumour entitieswhich range from potent apoptosis induc-tion33 34 to antiapototic protection21 35 or IFN-ãresistance.10 15 Moreover, the molecular deter-minants of either apoptosis or cell cycle arrestas alternative biological outcomes of IFN-ãtreatment are poorly understood and likelyintimately connected to the tumour specific setof oncogenic alterations. As defects in G1 cellcycle checkpoint control and/or apoptoticpathways contribute to a varying extent to thetransformed phenotype of individual tumours,it has proved diYcult to predict IFN-ã eVectson a given tumour cell. Accordingly, the thera-peutic potential of IFN-ã so far remains to beevaluated for each tumour entity individually.

Pancreatic cancer is characterised by defec-tive G1 control as a result of loss of p16 anddeficiency of apoptotic pathways due to loss ofthe tumour suppressor p53.36 Additional de-fects of apoptotic pathways appear likely inview of the fact that pancreatic cancer cells areresistant to a broad variety of proapototicstimuli, such as most chemotherapeutic ap-proaches and radiation therapy.37 As a conse-quence, patients with advanced non-resectablepancreatic cancer face a dismal prognosis withmedian life expectancy measured in the rangeof 4–6 months, which ranks pancreatic cancerfifth as a cause of cancer related deaths inWestern countries.38

Thus an improved understanding of theprevalent defects in cell cycle control andapoptosis signalling as well as detection ofintact proapoptotic pathways may help todelineate new improved strategies in the treat-ment of pancreatic cancer. In view of the clini-cal availability and known antiproliferative andproapototic potential of IFN-ã, we tested theactions of this cytokine in a representativepanel of human ductal pancreatic carcinomacell lines and defined the growth regulatorymechanisms.

Materials and methodsMATERIALS

Dulbecco’s modified eagle medium, RPMI1640 medium, phosphate buVered saline(PBS), and Lipofectamine transfection reagentwere purchased from Gibco BRL (Berlin, Ger-many). Fetal calf serum (FCS), trypsin/EDTA,penicillin, and streptomycin were from Bio-chrom (Berlin, Germany). Recombinant humanIFN-ã and the Cell Death Detection ELISAwere obtained from Boehringer Mannheim(Mannheim, Germany). Z-vad-fmk and theanti-poly(ADP ribose) polymerase (PARP)antibody were purchased from Calbiochem/Oncogene Research Products (Bad Soden,Germany). Other primary antibodies werefrom Santa Cruz Corporation (Santa Cruz,California, USA) and secondary antibodiesfrom Dianova (Hamburg, Germany). Reagentsfor western analysis were from BioRad Labora-tories (Munich, Germany), except for proteinA-sepharose beads which were from SigmaChemical Co (Deisenhofen, Germany), poly-vinyl difluoride (PVDF) membranes from NEN(Köln, Germany), and nitrocellulose mem-branes from Pharmacia Biotech (Freiburg,Germany). Reverse transcriptase-polymerasechain reaction (RT-PCR) reagents were fromPromega (Heidelberg, Germany). All otherreagents were obtained from Merck (Darm-stadt, Germany).

CELL CULTURE

Human pancreatic carcinoma cell lines AsPc-1,Capan-1, and Capan-2 were obtained from theAmerican Type Tissue Culture Collection.Dan-G cells were provided by the DeutschesKrebsforschungszentrum (Heidelberg, Ger-many). AsPc-1, Capan-1, and Dan-G cells weregrown as subconfluent monolayer cultures inRPMI 1640 medium supplemented with FCS(AsPc-1, 20%; Capan-1, 15%; Dan-G, 10%),and Capan-2 cells in Dulbecco’s modified eaglemedium supplemented with 10% FCS. Allmedia contained 100 U/ml penicillin/streptomycin and 2 mM L-glutamine. Cells weremaintained in 95% air and 5% CO2 at 37°C.Experiments were carried out in the log phase ofgrowth after cells had been allowed to attachovernight.

RNA PREPARATION AND RT-PCR

Total RNA was prepared using RNAzol R rea-gent (WAK Chemie, Bad Soden, Germany)according to the manufacturer’s instructions.RNA was submitted to DNAse digestion andaliquots of 1 µg were used for reversetranscription using Moloney murine leukaemiavirus RT. The following 5'-primers and 3'-primers were designed complementary to thenucleotide sequence of the human IFN-ã-receptor á-chain: 5'-ACG-CAGAAGGAAGATGATTGTGACG-3' and 5'-TCTATTGGAGTCAGATGGCTG-CCC-3'. The expectedsize of the PCR amplificate was 559 bp. Thereaction was carried out in 10 mM Tris HClbuVer (pH 8.3) containing 50 mM KCl, 0.01%Triton X 100, 1 mM MgCl2, 200 µM of eachdNTP, 50 µM of each primer, and 2.5 U ofThermus aquaticus DNA polymerase in a final

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volume of 50 µl. Amplification conditions for35 cycles were as follows: denaturation for 30seconds at 92°C, annealing at 60°C for 90 sec-onds, and extension for 90 seconds at 72°C. Anadditional 10 minute extension period wasadded to the final cycle.

IMMUNOPRECIPITATION AND IMMUNOBLOTTING

Approximately 5×106 cells were washed twicewith PBS and lysed in 1 ml of immunoprecipita-tion buVer (20 mM Tris (pH 7.8), 150 mMNaCl, 2 mM EDTA, 50 mM â-glycerophos-phate, 0.5% TNP 40, 1% glycerol, 1 mMNa3Vo4, 1 mM DTT, 10 IU/ml aprotinin, 10mM NaF, 2 µM leupeptin, and 2 mMphenylmethylsulphonylfluoride). Lysates weresonicated and left at 4°C for 30 minutesfollowed by centrifugation at 15 000 g for 10minutes. Aliquots of 1 mg of protein were thenpre-cleared for one hour with proteinA-Sepharose beads. Immunocomplexes werecollected on protein A-Sepharose coated withsaturating amounts of antibodies to Jak-1, Jak-2,and Stat-1, respectively. Beads were subse-quently washed five times with ice coldimmunoprecipitation buVer, boiled in Laemmlisample buVer, separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis(SDS-PAGE), and transferred to nitrocellulosemembranes. Non-specific binding was blockedwith wash buVer (10 mM Tris HCl, pH 7.6, 150mM NaCl, EDTA, 0.05% Tween 20) contain-ing bovine serum albumin (1%) and ovalbumin(2%) for two hours at room temperature. Incu-bation with the antiphosphotyrosine antibody (1µg/ml) was carried out overnight at 4°C and wasfollowed by three washes in wash buVer at roomtemperature. After incubation with goat anti-mouse horseradish peroxidase conjugated sec-ondary antibodies, membranes were washed3×10 minutes in wash buVer and bands werevisualised by enhanced chemiluminescence(ECL; Amersham). Membranes were strippedin a solution of 62.5 mM Tris HCl, pH 6.8, 2%SDS, and 100 mM â-mercaptoethanol for 30minutes at 55°C and resubmitted to immuno-blotting with the appropriate concentration ofprimary antibody.

For analysis of Rb, pro-Caspase-1, andinterferon regulatory factor 1 (IRF-1), 20 µgaliquots of the above lysates were submitted toSDS-PAGE and blotted onto PVDF mem-branes. Immunodetection was carried outessentially as described, except that 5% non-fatdry milk in PBS supplemented with 0.1%Tween (PBST) was used for blocking andPBST for all washing procedures.

TRANSIENT TRANSACTIVATION ASSAYS

Cells were plated in 35 mm culture dishes atapproximately 60% confluency. Transienttransfections were then carried out for 18hours in Ultraculture medium (Bio Whittaker,Walkersville, Maryland, USA) utilising 10 µl oflipofectamine and 1.5 µg of the GAS-luciferaseconstruct per well. Cells were allowed torecover for six hours in FCS supplementedmedium followed by stimulation with IFN-ã inUltraculture medium. After cell lysis, luciferaseactivity was determined using luciferin, ATP,

and coenzyme A (Promega Systems, Man-nheim, Germany), as recommended by themanufacturer.

GROWTH ASSAYS

Anchorage dependent growthCells were plated in 24 well culture dishes at adensity of 15 000 cells/well and vehicle orIFN-ã was added. At the indicated times, cellswere washed with PBS and harvested bytrypsinisation. Viable cells were identified bytrypan blue exclusion and counted in a haemo-cytometer. Triplicate wells were analysed foreach condition.

Anchorage independent growthClonal growth of pancreatic tumour cells wasexamined based on colony formation in agarsuspension, exactly as previously described.39

Briefly, 103 cells were plated as a single cell sus-pension in methylcellulose and incubated over10 days. Colonies were scored microscopicallywith an arbitrary cut oV set at 30 cellsminimum.

FLOW CYTOMETRY

A total of 106 cells per condition were washedin PBS and fixed in 70% ethanol at 4°C for onehour followed by resuspension in 500 µl ofPBS. After addition of 10 µl RNase (10mg/ml), cells were left for 30 minutes at 37°Cand stained with 10 µl propidium iodide (1mg/ml). Cellular DNA content was deter-mined for 10 000 cells on a FACScan utilising“Cellquest” software (Becton Dickensen, Hei-delberg). Cells with subdiploid DNA contentwere quantitated.

DETERMINATION OF APOPTOSIS

Cells were plated in 15 cm dishes and treatedas indicated. Floating and adherent cells wereharvested, combined, and counted to be usedin apoptosis assays as follows.

Cleavage of poly (ADP-ribose) polymerase(PARP)Aliquots of 106 cells were lysed in 250 µl ofbuVer containing 62.5 mM Tris/HCl (pH 6.8),6 M urea, 10% glycerol, 2% SDS, 0.00125%bromophenol blue, and 5% â-mercaptoethanol.After sonication for 15 seconds and incubationat 65°C for 15 minutes, lysates were separatedby SDS-PAGE. PARP immunodetection wascarried out as described above using theanti-PARP antibody at a dilution of 1:1000.

DNA fragmentationA total of 106 cells were digested at 37°C in300 µl of lysis buVer (10 mM Tris HCl (pH8.2), 400 mM NaCl, 2 mM EDTA, 1% SDS,and 300 µg/ml protease K). An equal volume of6 M NaCl was added and samples were vigor-ously vortexed. After brief centrifugation at2000 g, nucleic acids were precipitated fromsupernatants by addition of two volumes ofethanol. Pellets were solved in TE (10 mMTris HCl (pH 7.5) and 0.2 mM EDTA)and incubated for 30 minutes at 37°C with20 µg/ml RNase. DNA was separated on a2% agarose gel.

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Cell Death Detection ELISACells were incubated in lysis buVer suppliedwith the Cell Death Detection ELISA plus(Boehringer Mannheim, Mannheim, Ger-many) and nuclei were separated from thecytosolic fraction by centrifugation (five min-utes, 2000 g). Cytoplasmic histone associatedDNA fragments were determined as describedby the manufacturer.

STATISTICAL ANALYSIS

Statistical analysis was performed by one wayANOVA using the Newman-Keuls test (Prism,Graph pad software, San Diego, California,USA). DiVerences were considered significantat p<0.01.

ResultsHUMAN PANCREATIC CANCER CELLS EXPRESS

FUNCTIONAL IFN-ã RECEPTORS THAT ACTIVATE

THE Jak-Stat SIGNALLING PATHWAY

The responsiveness of tumour cells to IFN-ãmay vary due to loss or functional inactivationof components of the IFN-ã signal transduc-tion pathway. Therefore, IFN-ã signalling was

investigated in a panel of human pancreaticcancer cell lines: AsPc-1, Capan-1, Capan-2,and Dan-G.

Initially, we examined expression of IFN-ãreceptors by RT-PCR. Using specific primersagainst the á-chain of the IFN-ã receptor, wedemonstrated PCR amplificates of the ex-pected size, confirming that IFN-ã receptormRNA transcripts were present in all cell lines.Additional PCR without prior reverse tran-scription did not reveal amplificates, excludinggenomic contamination (fig 1).

To confirm that expression of IFN-ã recep-tor mRNA translates into functionally compe-tent surface receptors, ligand initiated signaltransduction was analysed next. Activation ofJak kinases by IFN-ã was examined onimmunoprecipitates of Jak-1 and Jak-2 usingantiphosphotyrosine antibodies (fig 2A, B; toppanel). Bands of 130 kDa, representing phos-phorylated Jak-1 and Jak-2 proteins, were firstapparent after one minute of IFN-ã stimulationand further increased at five minutes. Althoughphosphotyrosine bands in Capan-2 cells wereweak compared with the other cell lines, amoderate increase in Jak1 and Jak2 phosphor-ylation was also observed in response to IFN-ãstimulation. Using an analogous immunopre-cipitation approach, increased Stat-1 tyrosinephosphorylation was evident at one, five, and15 minutes (fig 2C; top panel). Both the 91kDa (Stat-1á) and the 84 kDa (Stat-1â) splicevariants were phosphorylated, albeit the rela-tive abundance of the 84 kDa form wasconsiderably lower and barely detectable inCapan-2 cells. To ensure that equivalentamounts of protein had been analysed in theimmunoprecipitations, immunoblots werereprobed with anti-Jak-1, anti-Jak-2, and anti-Stat-1 antibodies, respectively (fig 2A, B, C;bottom panels).

Figure 1 Pancreatic cancer cells express interferon ãreceptor (IFN-ã-R) mRNA transcripts. Total mRNA wasextracted, reverse transcribed into cDNA, and amplifiedusing polymerase chain reaction with specific primersdirected against the IFN-ã-R á chain. Alternating lanesrepresent reaction with (+) or without (−) addition ofreverse transcriptase (RT). The expected size of theamplification product was 559 bp and a 100 bp DNAstandard was included for size determination (lane 1).

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Figure 2 Interferon ã (IFN-ã) activates the Jak-Stat signal transduction pathway in human pancreatic cancer cells. Cellswere stimulated with 500 IU/ml IFN-ã for the indicated time periods and Jak-1 (A), Jak-2 (B), and Stat-1 (C) wereimmunoprecipitated (IP) from cell lysates. Immunoblots were performed to determine IFN-ã induced changes in thephosphotyrosine content of Jak and Stat complexes (top panels). To ensure that equal amounts of protein had beenexamined, the western blots (WB) were subsequently stripped and reprobed with Jak-1, Jak-2, or Stat-1 antibody,respectively (bottom panels). Molecular masses were deduced from a molecular size marker electrophoresed in parallel.

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To verify that activation of the Jak-Stat path-way results in activation of IFN-ã regulatedgenes, transactivation of a GAS-luciferasereporter construct was evaluated in transienttransfection assays. AsPc-1 cells were used as arepresentative cell line in these experiments aswe repeatedly failed to achieve acceptable tran-sient expression levels of the reporter constructin the other cell lines. IFN-ã treatment dosedependently induced luciferase activity (fig 3),indicating that IFN-ã was capable of stimulat-ing GAS dependent gene expression in humanpancreatic cancer cells.

IFN-ã INHIBITS ANCHORAGE DEPENDENT AND

ANCHORAGE INDEPENDENT GROWTH OF HUMAN

PANCREATIC CANCER CELLS

Having established functional IFN-ã signallingin the cell lines investigated, we evaluated theeVects of IFN-ã on pancreatic cancer cellgrowth. To examine anchorage dependentgrowth, cell numbers were determined over aperiod of four days. During this time, controlcells grew exponentially whereas proliferationof IFN-ã treated cultures (500 IU/ml) was pro-foundly reduced in all four cell lines (fig 4A).Growth inhibition was notably absent duringthe first two days of IFN-ã treatment butbecame apparent on day three in all cell lines.However, once growth inhibition was detect-able, two cell lines (Capan-1 and Capan-2)presented a complete block in proliferation.

To determine if growth inhibition could beachieved with therapeutically relevant concen-trations of IFN-ã, a dose-response relation wasestablished after four days of IFN-ã treatment.The antiproliferative action of IFN-ã was dosedependent in all four cell lines although sensi-tivity to IFN-ã varied with EC50 values below10 IU/ml in AsPc-1 and approximately 50IU/ml in Dan-G cells (fig 4B). Despite modestJak/Stat1 activation, proliferation of Capan 2cells was strongly inhibited (15.7 (2.3)% ofcontrol; EC50 32.6 (1.1) IU/ml).

Because growth of malignant tumours invivo is influenced by unique properties oftransformed cells more accurately examined inanchorage independent growth assays, colony

Figure 3 Interferon ã (IFN-ã) stimulation results intransactivation of a GAS driven reporter construct. AsPc-1cells were transiently transfected with pGL2-GAS andrelative luciferase activity was measured after a 24 hourperiod of IFN-ã stimulation with the indicated doses. Datarepresent mean (SEM) values from at least three separateexperiments conducted in triplicate.

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Figure 4 Interferon ã (IFN-ã) inhibits anchorage dependent and independent growth of human pancreatic cancer cells.Subconfluent cells were treated with 500 IU/ml IFN-ã or vehicle for the indicated time periods (A), or with the indicatedfinal concentrations of IFN-ã for 96 hours (B) and cell numbers were determined. (C) A single cell agar suspensioncontaining 1×103 cells was incubated with the indicated doses of IFN-ã for 10 days and colony formation was evaluated.Data represent mean (SEM) values from at least three separate experiments, each conducted in triplicate (*p<0.01).

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formation in agar suspension was also evalu-ated. IFN-ã treatment profoundly reduced thenumber of colonies in the pancreatic cancercell lines studied (fig 4C), except for Dan-Gcells which do not form colonies. Again,growth inhibition was dose dependent withmaximal decreases in colony number to 25.3(0.6)% of control in AsPc-1 and 24.7 (3.2)% inCapan-2 achieved with 1000 IU/ml and to 35.2(8.7) in Capan-1 cells with 500 IU/ml IFN-ã.

THE ANTIPROLIFERATIVE ACTION OF IFN-ã INPANCREATIC CANCER CELLS IS DUE TOINDUCTION OF APOPTOSIS

To identify potential mechanisms of IFN-ãmediated growth inhibition, cell cycle distribu-tion was monitored by flow cytometry (fig 5).During a four day period the fraction of cells inthe individual cell cycle phases did not changein untreated controls. In contrast, IFN-ãtreated cultures revealed an increased percent-age of cells with subdiploid DNA content overtime, suggesting induction of apoptosis (fig5A). A quantitative analysis of the DNA histo-grams confirmed a substantial amount of cells

with subdiploid DNA content in IFN-ã treatedcultures with percentages ranging from 19.7(3.1)% in AsPc-1 to 47.3 (5.8)% in Capan-1cells after 96 hours of IFN-ã stimulation (fig5B). In good agreement with the kinetics ofgrowth inhibition observed in substrate de-pendent proliferation assays (correlation coef-ficient r=0.9892 for Capan-1) this putativepopulation of apoptotic cells was first observedafter two days and then dramatically increasedwith prolonged treatment (fig 5A, B). Also, theextent and dose dependence of substratedependent growth inhibition and the appear-ance of the pre-G1 population were almostidentical (r=0.9563 for Capan-1), suggestingthat apoptosis might fully account for thereduction in cell numbers (fig 5C).

As the pre-G1 peak described above does notprovide conclusive evidence of apoptosis, weperformed experiments to corroborate IFN-ãmediated apoptosis. Firstly, DNA integrity wasevaluated after four days. Whereas IFN-ãtreated cultures of all four cell lines presentedthe typical DNA laddering, essentially no DNA

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Figure 5 Interferon ã (IFN-ã) treated pancreatic tumourcells contain a subdiploid DNA complement. (A) Timecourse of representative FACS analyses illustrating the cellcycle distribution of pancreatic cancer cells under controlconditions (top panel) or in the presence of 500 IU/mlIFN-ã (bottom panel). (B) Summary of IFN-ã inducedcell cycle redistribution. Mean (SEM) percentage of cellswith subdiploid DNA content were determined in threeindependent experiments. *p<0.01 versus time matcheduntreated controls. (C) Alignment of growth inhibition andapoptosis.


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degradation was observed in untreated AsPc-1and Capan-2 cells (fig 6A). Corresponding totheir relatively high rates of spontaneous apop-tosis (see also fig 5), Capan-1 and Dan-G cellsrevealed faint DNA laddering under controlconditions, which was however greatly en-hanced by IFN-ã treatment.

DNA fragmentation was further confirmedand quantitated in a complementary approachusing a histone ELISA with antibodies directedagainst the DNA or the histone component ofthe nucleosomes, respectively. Starting at 24hours, cytoplasmic extracts of IFN-ã treatedcells revealed a distinct increase in DNA-histone complexes compared with control cul-tures (fig 6B). Thus the onset of DNAfragmentation preceded the appearance of thepre-G1 population observed in the FACSanalyses, most likely reflecting more sensitivedetection of cells that initiate DNA fragmenta-tion in the histone ELISA.

PARP is an early target of active caspases inapoptosis and the resulting 85 kDa cleavageproduct may be used as a separate monitor ofapoptosis. In immunoblot analyses of controlcultures, PARP was detected nearly exclusivelyas a 116 kDa band, representing the intactPARP protein. In contrast, a prominent 85 kDa

band was time dependently induced in IFN-ãtreated cells (fig 6C). Again, the first eVectswere noted after 24 hours of IFN-ã treatment.Similar to the DNA fragmentation assay, fewindividual control cultures also revealed a weakbut distinct 85 kDa fragment indicative ofspontaneous apoptosis under tissue cultureconditions.

IFN-ã-MEDIATED APOPTOSIS DOES NOT REQUIRE

pRb REGULATION

In response to IFN-ã, pRb appears to functionas a determinant of G1 cell cycle arrest and asa caspase substrate. Therefore, IFN-ã eVectson cellular content and phosphorylation statusof pRb were examined (fig 7). Under controlconditions, pRb was detected primarily in itshyperphosphorylated growth permissive form.In none of the four cell lines did IFN-ã aVecteither expression or phosphorylation of pRbwithin the first 12 hours. At later time points,AsPc-1 and Capan-1 displayed marked hypo-phosphorylation and a distinct reduction ofoverall protein content consistent with apop-totic degradation. In contrast, no alteration inpRb was observed in Capan-2 or Dan-G cells.Thus IFN-ã mediated apoptosis of pancreaticcancer cells appeared to be independent of

Figure 6 Interferon ã (IFN-ã) induced DNA fragmentation and poly(ADP ribose) polymerase (PARP) cleavage inhuman pancreatic cancer cell lines. (A) Cells were incubated for four days with 500 IU/ml IFN-ã (lanes 3, 5, 7, and 9) orleft untreated (lanes 2, 4, 6, and 8) and genomic DNA was subsequently analysed for oligonucleosomal fragmentation. Forsize determination a 100 bp DNA standard was used (lane 1). (B) Cells were treated with vehicle or IFN-ã (500 IU/ml)for the indicated time periods and examined using a specific histone DNA ELISA to quantitate DNA fragmentation. Mean(SEM) absorbance at 405 nm relative to untreated controls was determined in three independent experiments, eachperformed in triplicate. (C) Immunoblot demonstrating expression of PARP (p 116) and its apoptosis related cleavageproduct p 85 in untreated controls and cells that had been treated with 500 IU/ml IFN-ã for the time periods indicated.Alternating lanes represent IFN-ã treated (+) and untreated (−) cells. Whole cell lysates were separated by 7.5% sodiumdodecyl sulphate-polyacrylamide gel electrophoresis.

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pRb. In addition, these data argue against apRb mediated G1 specific cell cycle inhibitionprior to the onset of apoptosis.

IFN-ã MEDIATED INDUCTION OF APOPTOSISINVOLVES UPREGULATION AND ACTIVATION OFTHE AMPLIFIER PROTEASE CASPASE-1

To evaluate the role of caspases in IFN-ãinduced apoptosis, we next tested the ability ofthe caspase inhibitor z-vad-fmk to preventapoptosis. Cells were treated with a combina-tion of IFN-ã and the caspase inhibitor, andapoptosis was quantitated by flow cytometryafter three days. Addition of z-vad-fmk alonedid not change cell cycle distribution (data notshown). However, the IFN-ã induced pre-G1peak was completely abrogated by the caspaseinhibitor in all four cell lines (fig 8A, B).Furthermore, prevention of apoptosis by

z-vad-fmk was dose dependent, as demon-strated in experiments on Capan-1 cells (fig8B).

Z-vad-fmk potentially inhibits caspases 1, 3,4, and 7. Of these, caspase-1 is unique in that itqualifies as an amplifier rather than anexecutioner caspase and is located most proxi-mal within the caspase cascade. Therefore,changes in caspase-1 were investigated inAsPc-1 and Dan-G cells as representative celllines. In view of the delayed onset of apoptosis,changes in caspase expression rather thanactivity were examined (fig 9A). In untreatedcontrols, weak bands of 45 kDa were detected,corresponding to procaspase-1. FollowingIFN-ã treatment, a time dependent distinctinduction of the 45 kDa band was observedstarting at six hours in AsPc1 and at 12 hoursin Dan-G cells, which then persisted through-out the observation period of 72 hours.Compared with their time matched controls,IFN-ã treated cells displayed not only in-creased abundance of the proform but alsobands of approximately 35 kDa (fig 9B),indicative of enzyme activation.40 As thecaspase-1 antibody used does not recognise the20 kDa mature processing product and the 10kDa subunit of the mature active complex haspreviously been shown to be highly instable,40

these smaller subunits were not detected.Studies on the regulation of procaspase-1

expression have identified various regulatorysites in the procaspase-1 promoter, includingan IRF-1 binding site. Therefore, eVects ofIFN-ã on IRF-1 expression were investigatedby immunoblotting (fig 9B). Bands of theappropriate size of 48 kDa were barely detect-able in either AsPc-1 or Dan-G controlcultures. Stimulation with IFN-ã resulted in aprofound increase in IRF-1 protein starting asearly as three hours. Subsequently, IRF-1 levelsremained elevated until the end of the observa-tion period at three days. Thus induction ofIRF-1 preceded the manifestation of apoptosis.Similar IRF-1 upregulation was observed inCapan-1 and Capan-2 cells (data not shown).

DiscussionInduction of apoptosis represents a paramountgoal in cancer therapy. In contrast with othercommon malignancies, pancreatic cancer ischaracterised by resistance to a broad spectrumof proapoptotic stimuli, such as chemotherapyand radiation.37 Although prevalent geneticalterations in pancreatic cancer have been speci-fied, the basis for the striking deficiency inapoptosis susceptibility remains to be eluci-dated.36 41 Altered expression of Bcl 2 familymembers is variably observed in histochemicalanalysis of pancreatic tumour specimens andstudies utilising permanent human pancreaticcancer cell lines, but the biological relevance ofthese observations has remained contro-versial.42–45 In addition, pancreatic cancer wassuggested to circumvent apoptosis mediated byimmunsurveillance mechanisms by means ofFas ligand mediated counter attack.46

Using a representative panel of human pan-creatic cancer cell lines, the current studyinvestigated the antiproliferative actions of

Figure 7 Interferon ã (IFN-ã) treatment regulates retinoblastoma protein (pRb)phosphorylation and abundance in human pancreatic cancer cells. Immunoblot analysisdemonstrating the time course of changes in pRb expression and phosphorylation status incontrol and IFN-ã stimulated human pancreatic cancer cells. In each lane, 20 µg of wholecell lysates were separated by 7.5% sodium dodecyl sulphate-polyacrylamide gelelectrophoresis. Shown is a representative of two experiments yielding similar results. pRb,ppRb, hypo- and hyperphosphorylated forms of Retinoblastoma protein, respectively.

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Figure 8 Interferon ã (IFN-ã) mediated apoptosis is prevented by the caspase inhibitorz-vad-fmk. Cells were stimulated for three days with either IFN-ã (500 IU/ml), acombination of IFN-ã and z-vad-fmk at various doses, or were left untreated.Subsequently, cells were analysed by flow cytometry and the fraction of cells with subdiploidDNA content was quantitated. Mean (SEM) percentage of cells with subdiploid DNAcontent was determined in three independent experiments.

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IFN-ã. Responsiveness to IFN-ã varies consid-erably between diVerent tumours and cell typesand IFN-ã resistance is common in sometumour entities—for example, in mesothe-lioma,47 ovarian,48 mammary,15 lung, andmelanoma cancer10 cells. This may be due toreduced IFN-ã receptor expression duringmalignant transformation, as has been sug-gested in endothelial cells,11 or to deficientdownstream components in the IFN-ã signal-ling pathway. A careful analysis of IFN-ãresistant lung tumour cell lines revealedfunctional inactivation of Jak2, loss of Jak-1,and loss of the IFN-ã receptor á chain asunderlying molecular defects.10 Similarly, acomparison of IFN-ã responsive or resistanthuman mesothelioma cell lines connected thefailure of IFN-ã to inhibit DNA synthesis withaltered expression or activation of Jak-2 orStat-1 and low induction of IRF-1, respec-tively.49 As tumour specific defects occur at allsteps of the IFN-ã signalling pathway, the indi-vidual signalling components were carefullyanalysed in the current study. Firstly, we dem-onstrated the presence of IFN-ã receptormRNA transcripts. Secondly, we documentedexpression of key eVectors of IFN-ã—that is,Jak-1, Jak-2, and Stat-1—in pancreatic cancercells and their activation in response to ligand

stimulation. Thirdly, transactivation of a GASdriven reporter construct and induction of anendogenous IFN-ã inducible gene product(IRF-1) were observed, establishing a function-ally intact IFN-ã signalling pathway in all pan-creatic cancer cell lines examined. Specifically,Stat-1 and IRF-1 downstream eVectors arewell established critical mediators of IFN-ãdependent growth inhibition50 51

When subsequently evaluated in anchoragedependent and independent growth assays,IFN-ã was indeed capable of profoundlyinhibiting proliferation of pancreatic cancercells. Remarkably, all four pancreatic cancercell lines were highly sensitive towards IFN-ãand a significant antiproliferative eVect wasobtained in the range 8–30 IU/ml IFN-ã— thatis, within the range of therapeutically achiev-able concentrations.52 The reasons for minordiVerences between individual cell lines re-garding sensitivity to and eYcacy of IFN-ãaction remain to be determined. Previousreports suggested that the extent or kinetics ofactivation of Jak-2 and Stat-1, respectively,accounted for diVerential responsiveness ofmesothelioma cell lines towards IFN-ã.49 Inour study, we noted a comparably low eYcacyof IFN-ã to induce activation of these eVectorsin Capan-2 cells. However, the strong induc-tion of IRF-1 that was consistently observed inall four pancreatic cell lines contradicts a linearrelation between Jak-Stat signalling and activa-tion of growth relevant eVectors. Furthermore,the growth inhibitory potential of IFN-ã inCapan-2 cells was well within the rangeobserved in the other cell lines and specificallyexceeded the antiproliferative action in AsPc-1cells.

IFN-ã treated pancreatic cancer cell culturestypically completed one round of replicationwithout perceivable changes in cell cycle distri-bution prior to the subsequent almost com-plete block of proliferation. Thus their re-sponse diVered from other cell models22 53 54

where a CKI mediated inhibition of cyclindependent kinase activity rapidly resulted inG1 arrest on the basis of pRb hypophosphor-ylation15 22 or pRb independent mechanisms.53

Of note, the lack of cell cycle redistribution didnot result from loss of pRb, as we documentedcontinued pRb expression in all cell lines.Instead of cell cycle arrest, the appearance ofsubdiploid cells in cell cycle analyses suggestedapoptosis induction, which was confirmed by(i) demonstration of DNA fragmentation, (ii)detection of the apoptotic 85 kDa PARP cleav-age product, and (iii) functional requirementfor caspase activity. As the dose-response rela-tionship of growth inhibition and induction ofapoptosis closely correlated, apoptosis prob-ably constituted the main if not the exclusivemechanism underlying IFN-ã mediatedgrowth inhibition.

The proapoptotic actions of IFN-ã were latein onset, suggesting that delayed signallingevents such as synthesis of intermediary prod-ucts were involved. Consistent with thishypothesis we found IFN-ã mediated induc-tion of caspase-1 prior to the onset of apopto-sis. Within the caspase cascade, caspase-1

Figure 9 Interferon ã (IFN-ã) causes upregulation of procaspase-1 and interferonregulatory factor 1 (IRF-1) in pancreatic cancer cell lines. (A) Cells were incubated withvehicle or IFN-ã (500 IU/ml) for the indicated time periods. Aliquots of 20 µg of whole celllysate were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS PAGE) and subsequently incubated with an anticaspase antibody that recognises adouble band of the procaspase precursor form at approximately 45 kDa. (B) Immunoblotrevealing caspase-1 processing intermediates with the 35 kDa form indicative of activation.Lysates were from AsPc-1 and Dan-G cells treated for 48 and 72 hours with 500 IU/mlIFN-ã or time matched control cells. In Dan-G cells, top and bottom panels represent lightand darker exposures of the same blot. (C) Immunoblot analysis for IRF-1 expression. Cellswere incubated with vehicle or IFN-ã (500 IU/ml) for the indicated time periods. In eachlane, 20 µg of whole cell lysates were separated by 12% SDS-PAGE and IRF-1 expressionwas determined using a monospecific antibody. Shown are representative blots of at leasttwo experiments.

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assumes an intermediary function between ini-tiator and executioner caspases and has beensuggested as an amplifier of proapoptoticsignalling.28 Proteolytic cleavage of the inactiveprocaspase-1 by upstream initiator caspases,such as caspase-8 or caspase-9, represents theprevalent mode of caspase-1 activation.28 29.However, autocatalytical cleavage followingsubstantial endogenous or exogenous upregu-lation of proenzyme expression may bypass therequirement for upstream activators.55 56 ThusIFN-ã mediated induction of procaspase-1 inpancreatic cancer cells may trigger its activa-tion and thereby initiate activation of down-stream executioner caspases such as caspase-3.An analogous IFN-ã mediated induction ofcaspase-1 has previously been reported inHeLa and A431 cells,55 vascular smoothmuscle cells,57 leukaemia cells,19 and Sertolicells.58 The functional relevance of caspase-1activation for apoptosis initiation in pancreaticcancer cells was underlined by the ability of thecaspase inhibitor z-vad-fmk to completely pre-vent IFN-ã eVects. This is in good agreementwith results from studies on primary spleno-cytes from caspase-1 (−/−) mice,55 providingconclusive evidence for the requirement ofcaspase-1 in IFN-ã dependent apoptosis inthese cells.

Analyses of the caspase-1 promoter haverevealed binding sites for IRF-1. IRF-1 itselfrepresents an immediate IFN-ã response prod-uct59 60 critically involved in IFN-ã mediatedapoptosis as IFN-ã induced caspase-1 activa-tion was completely abrogated in IRF-1deficient mice.59 Furthermore, the growthinhibitory action of IFN-ã in KG1 humanmyelocytic leukaemia cells could be convertedinto a proliferative stimulus by retroviralexpression of IRF-1 antisense.61 62 Also, resist-ance to interferon induced apoptosis was asso-ciated with IRF-1 gene rearrangements63 ordefective IRF-1 binding to the caspase-1promotor64 in HCC cell lines or Daudi cells. Inpancreatic cancer cells, we demonstrated sus-tained induction of IRF-1 protein content,consistent with IRF-1 constituting the func-tional link between IFN-ã initiated signallingevents and upregulation of procaspase-1. Inparticular, the time course of IRF-1 inductionplaced this event between early activation ofStat-1 and delayed induction of procaspase-1.

Once procaspase-1 has been induced andconverted into the active caspase, proteolyticactivation of downstream eVector caspasesresults. Caspase-3 represents one of thebiologically most relevant downstream targetsof caspase-1 as it functions to incapacitate ordestroy essential homeostatic pathways duringthe eVector phase of apoptosis. Substratesinclude endonuclease inhibitors,65 PARP,66 67

and most likely pRb.68 69 70 Consistent with thecaspase-1 dependent activation of caspase-3described in the literature,28 we were able todetect oligonucleosomal DNA fragmentation,PARP cleavage, and pRb downregulation inIFN-ã-treated pancreatic cancer cells. Thereduction in pRb protein levels was precededby a shift from the hyper to its hypophosphor-ylated form. Similar regulation of pRb during

apoptosis has been extensively characterised inhuman leukaemia cell lines,71 where it resultedfrom dephosphorylation and subsequent deg-radation triggered by an ICE-like protease.Given the established antiapoptotic function ofpRb, this caspase mediated degradation wasproposed as an important intermediate step inthe execution of apoptosis. A role for pRb deg-radation in the context of IFN-ã inducedapoptosis is supported by the observation thatreconstitution of pRb prevented apoptosis inIFN-ã treated pRb deficient bladder carcinomacells.72 In the current study however only two offour cell lines investigated presented appreci-able pRb reduction although they uniformlyunderwent apoptotic cells death, indicatingthat IFN-ã mediated apoptosis of pancreaticcancer cells can occur independent of pRbdegradation.

Other investigators have identified the dou-ble stranded RNA dependent protein kinasePKR as a critical parameter of IFN mediatedapoptosis induction.24 73 74 However, PKR in-duction occurs in response to both type I andtype II interferons. As IFN-á treatment wasineVective in inducing apoptosis in pancreaticcancer cell lines75 but resulted in S phase cellcycle delay (unpublished observation), a deci-sive role of PKR induction for IFN-ã mediatedapoptosis versus cell cycle eVects appearsunlikely. None the less, PKR induction may bea necessary intermediate step for either cellfate, as was demonstrated for IFN-á dependentG1 arrest in myeloid leukaemia cells,76 as wellas for IFN-ã dependent IRF-1 induction andstress induced apoptosis in mouse embryofibroblasts of PKR(−/−) mice.24

Based on both clinical experience and invitro data, the relative resistance of pancreaticcancer cells to conventional chemotherapyand/or radiation as well as to many biothera-peutic modalities is a disappointing althoughwell known fact. The tremendous increase inour understanding of the regulation andexecution of apoptosis has produced a consid-erable and successful eVort in revealing andelucidating molecular changes that are respon-sible for this “apoptosis resistance”. In accord-ance with this, the current study delineated anintact proapoptotic pathway in pancreatic can-cer cells which was found to be activated inresponse to the clinically available cytokineIFN-ã. Specifically, IRF-1 and/or procaspase-1may represent potential therapeutic targets tobe further explored in the treatment of pancre-atic cancer. Furthermore, IFN-ã should beevaluated in in vivo studies in animal models ofpancreatic cancer such that a valid basis forconsidering its clinical use is obtained.

SR was supported by a grant from the Deutsche Forschungsge-meinschaft (Ro674/13-1), Berliner Krebsgesellschaft, DeutscheKrebshilfe, Wilhelm Sander Stiftung, and Sonnenfeld-Stiftung.K Farwig was supported by the Sonnenfeld-Stiftung.

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1st Asia Pacific Forum on Quality Improvement in Health Care

Three day conference

Wednesday 19 to Friday 21 September 2001

Sydney, Australia

We are delighted to announce this forthcoming conference in Sydney. Authors are invited tosubmit papers (call for papers closes on Friday 6 April), and delegate enquiries are welcome.

The themes of the Forum are:

x Improving patient safetyx Leadership for improvementx Consumers driving changex Building capacity for change: measurement, education and human resourcesx The context: incentives and barriers for changex Improving health systemsx The evidence and scientific basis for quality improvement.

Presented to you by the BMJ Publishing Group (London, UK) and Institute for HealthcareImprovement (Boston, USA), with the support of the the Commonwealth Department ofHealth and Aged Care (Australia), Safety and Quality Council (Australia), NSW Health(Australia), and Ministry of Health (New Zealand).

For more information contact: [email protected] or fax +44 (0)20 7383 6869


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Interferon ª inhibits growth of human pancreatic carcinoma ...pressor interferon regulatory factor 1 (IRF-1). IFN-ª treatment profoundly in-hibited anchorage dependent and inde-pendent