Interferon-a Delays S-Phase Progression in Human HepatocellularCarcinoma Cells Via Inhibition of Specific

Cyclin-Dependent Kinases

DEREK MURPHY, KATHARINA M. DETJEN, MARTINA WELZEL, BERTRAM WIEDENMANN, AND STEFAN ROSEWICZ

The potential antiproliferative effects of interferon-a(IFN-a) in the treatment of hepatocellular carcinoma (HCC)are controversial, and the growth inhibitory mechanismsremain poorly understood. Therefore, the current study wasdesigned to delineate the molecular mechanisms responsi-ble for direct antiproliferative actions of IFN-a in HCC cells.IFN-a receptor expression and signal transduction were ex-amined by RT-PCR, immunoprecipitation, Western analy-sis, and transient transactivation assays. Effects of IFN-a oncell growth and cell-cycle distribution were evaluated basedon cell numbers and flow cytometry. Composition and ac-tivity of cyclin-dependent kinase complexes were deter-mined by immunoblotting and histone-H1-kinase assays.Expression of IFN-a receptors was found in all 3 HCC celllines. IFN-a binding initiated phosphorylation of Jak1 andTyk2 kinases leading to Stat1/Stat2 activation, nucleartranslocation, and transactivation of an ISRE-luciferase re-porter gene construct. IFN-a treatment resulted in a time-and dose-dependent reduction of proliferation. Cell cycleanalysis of G1-synchronized, IFN-a–treated HCC cells re-vealed a substantial delay in S-phase progression but noalteration of G1/S-phase transition or evidence of apoptoticcell death. Reflecting the time course of S-phase accumula-tion, cell cycle-dependent induction of Cyclin A and CyclinB was impaired, resulting in reduced activity of Cdk2 andCdc2 kinases. Furthermore, Cdc25C was selectively down-regulated. IFN-a treatment inhibits growth of HCC cells byspecifically delaying S-phase progression, most likely be-cause of inhibition of Cyclin A induction, resulting in de-creased activity of the associated Cdk2 and Cdc2 kinases.(HEPATOLOGY 2001;33:346-356.)

Hepatocellular carcinoma (HCC) is one of the most fre-quent malignancies worldwide. HCC commonly arises in livertissue damaged by long standing chronic inflammation. Fac-tors able to induce and maintain a chronic injury are diverseand include hepatitis B virus (HBV) and hepatitis C virus(HCV) infection1,2 as well as toxic agents such as alcohol3 andaflatoxins.4 Accordingly, a broad variety of genetic alterationscontribute to a multistep transformation process leading toclinical manifestation of HCC. The molecular targets of HCCcarcinogenesis encompass growth factors and their receptors,signaling cascades and effectors, as well as oncogenes, tumorsuppressors, and factors controlling matrix interaction andangiogenesis.5,6 Although several common alterations such asloss of the tumor suppressors p167 and p538,9 have been re-ported, tissue-specific molecular defects in HCC remainedelusive and thus inaccessible to therapeutic strategies.

Despite extensive clinical trials, the prognosis for patientswith advanced HCC has remained poor unless curative sur-gery can be performed.10 Because in most cases the disease isnot amenable to surgical resection at the time of diagnosis, anurgent need for systemic treatment is obvious.

Interferon-a (IFN-a) belongs to the interferon family ofcytokines originally identified because of their function in theantiviral host-defense.11 Further characterization of IFN-amediated biological effects revealed a prominent role in thecontrol of cellular proliferation and survival as well as potentimmunomodulatory and antiangiogenic actions. Therefore,IFN-a has been successfully included in treatment protocolsfor malignancies such as hairy-cell leukemia, chronic myelog-enous leukemia, and Kaposi’s sarcoma.12

IFN-a has proven effective in eliminating HBV and HCV ina considerable number of patients with chronic hepatitis.13,14

Furthermore, IFN-a receptors are present and functional inthe inflamed or cirrhotic liver tissue before transforma-tion.15,16 Accordingly, considerable expectations to reducethe incidence of HCC were connected with the use of IFN-a inantiviral treatment of hepatitis B or C. By now, first clinicaltrials have indeed confirmed a reduced incidence of HCC inIFN-a treated patients with chronic active hepatitis C-associ-ated cirrhosis.17-21 In contrast, the benefit derived from IFN-atreatment of established HCC remains controversial.22,23

Research over the past decade has comprehensively delin-eated the signal transduction pathways initiated in responseto IFN-a. IFN-a binds to specific type I IFN cell-surface re-ceptors.24 Binding of IFN-a to the receptor causes activationof the receptor associated tyrosine kinases, Jak1 and Tyk2,which in turn recruit and activate the transcription factorsStat1 and Stat2 via tyrosine phosphorylation. On het-

Abbreviations: HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitisC virus; IFN-a, interferon-alfa; DMEM, Dulbecco’s modified Eagle medium; PBS, phos-phate buffered saline; FCS, fetal calf serum; RT-PCR, reverse transcription-polymerasechain reaction; ISRE, interferon-stimulated response element; CMV, cytomegalovirus;SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; ECL, enhancedchemiluminescence.

From Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Uni-versitatsklinikum Charite, Campus Virchow-Klinikum, Humboldt Universitat zu Berlin.

Received August 8, 2000; accepted November 16, 2000.Supported by the Deutsche Forschungsgemeinschaft, Mildred-Scheel Stiftung and

Berliner Krebsgesellschaft (all to SR).Derek Murphy and Katharina M. Detjen contributed equally to this work.Address reprint requests to: Professor Dr. med. Stefan Rosewicz, Medizinische Klinik

mit Schwerpunkt Hepatologie und Gastroenterologie, Augustenburger Platz 13353, Ber-lin, Germany. E-mail: stefan.rosewicz@charite.de; fax: (49) 30-450-53-940.

Copyright © 2001 by the American Association for the Study of Liver Diseases.0270-9139/01/3302-0006$35.00/0doi:10.1053/jhep.2001.21749

346

erodimerization Stat1/Stat2 then translocate into the nucleus,where they control the expression of IFN-regulated genes orgene networks.25

Unlike these early well defined signaling events, growthregulatory pathways responsible for the antitumor action ofIFN-a are not well delineated and appear less conserved. Thismay in part result from the fact that multiple pathways con-tribute to the overall growth regulatory effects.26 Typically,direct antiproliferative action, combined with indirect effectssuch as immunmodulation and interference with angiogene-sis, participate in the biological outcome of IFN-a treatmentand may therefore considerably vary between different tumorentities. Even when focusing on the direct antiproliferativeactions of IFN-a in transformed cell models, various mecha-nisms have been observed. IFN-a has been described to altercell cycle progression by inducing a G0/G1 arrest, prolongingS-phase transition or arresting cells in G2/M.27-29 Further-more, induction of apoptosis has been reported as an alterna-tive response to IFN-a treatment.30-32 The variability in thesedirect responses may be influenced by cell type specific differ-ences in STAT complement and availability of coactivators.33

Alternatively, it reflects differences in the available spectrumof IFN-a–susceptible growth relevant effector molecules be-cause of specific oncogenic alterations present in the targetcell.

In an etiologically heterogeneous tumor entity such asHCC, a detailed molecular knowledge of the antiproliferativemechanisms appears crucial to critically redefine its potentialtherapeutic benefit. Importantly, delineating the componentsand targets of IFN-a growth-modulatory pathways should en-hance our understanding of its potential role and significancein HCC treatment. Therefore, the current study analyzed theaction of IFN-a in a panel of 3 human HCC cell lines. Specif-ically, the effects of IFN-a on central cell cycle regulatoryproteins were examined in detail in order to characterize thegrowth regulatory mechanisms and to identify relevant IFN-a-modulated target molecules in HCC.

MATERIALS AND METHODS

Materials. HepG2 and HuH7 cell lines were obtained from theATCC; SK-Hep-1 cells were a kind gift from D. Schuppan. Dulbecco’smodified Eagle medium (DMEM), RPMI 1640 medium and phos-phate buffered saline (PBS) were purchased from Gibco BRL (Berlin,Germany). Fetal calf serum (FCS), Trypsin/EDTA, penicillin, andstreptomycin were from Seromed (Berlin, Germany). The antibodiesfor Cdk2, Cdc2, Cdc25A, Cdc25C, Jak1, Tyk2, Stat1, and Stat2 werefrom Santa Cruz Biochemicals (Santa Cruz, CA), those for Cyclin A,Cyclin E, and Cyclin B were from Pharmingen (San Diego, CA) andthe a-phosphotyrosine antibody was from Transduction Laborato-ries (Lexington, KY). All secondary antibodies were purchased fomDianova GmbH (Hamburg, Germany). (g-32P)ATP was from Amer-sham (Braunschweig, Germany). Calf histone H1, DNase and ATPwere purchased from Boehringer Mannheim (Mannheim, Germany).Reagents for Western analysis were from BioRad Laboratories GmbH(Munich, Germany) and polyvinyl difluoride (PVDF)-membranesfrom NEN (Brussels, Belgium). IFN-a 2b (Roferon) was kindly sup-plied by Hofmann-LaRoche (Basel, Switzerland). PCR reagents werefrom Promega (Heidelberg, Germany). Protein A-sepharose beadsand all other reagents were from Sigma Chemical Co. (Deisenhofen,Germany).

Cell Lines and Culture. The HCC cell lines HepG2 and HuH7 weregrown in a culture medium consisting of Dulbecco’s modified Eaglemedium supplemented with 10% FCS, 100 U/mL penicillin, 100mg/mL streptomycin, and 2 mmol/L L-glutamine. SK-Hep-1 cellswere grown in RPMI 1640 medium supplemented with 20% FCS,

100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mmol/L L-glutamine. Growth conditions were 37°C in a humidified atmo-sphere of 5% CO2. Cells were cultured for 16 hours before the ex-periments to allow the cells to enter their logarithmic growth phase.

Anchorage Dependent Growth Assays. Cells were grown in 96 wellculture dishes at a density of 2,000 cells/well. After an attachmentperiod of 16 hours, IFN-a was added at the indicated concentration(10-103 IU/mL). Control cells received vehicle only (PBS). After theincubation period cells were trypsinated and stained with trypanblue. Viable cells were then counted using a hemocytometer. Tripli-cate wells were analyzed for each time point and IFN-a dosage.

Flow Cytometry. After trypsination, approximately 106 cells werecollected by centrifugation at 1,000g for 5 minutes. Cells were thenwashed in PBS followed by resuspension and fixation in 70% ethanolfor approximately 2 hours. Next, cells were briefly centrifuged,washed in PBS, and resuspended in 500 mL PBS containing 100 mgRNase and incubated for 30 minutes. Cellular DNA was then stainedby the addition of 10 mg propidium iodide, and a total of 10,000cells/condition were analyzed on a FACScan utilizing Cellquest soft-ware (Becton Dickinson).

Analysis of IFN-a Receptor Expression Using Reverse-Transcription Poly-merase Chain Reaction. Total RNA was prepared using RNAzol R re-agent (WAK Chemie, Bad Soden, Germany) according to the manu-facturer’s instructions. RNA was submitted to DNAse digestion, andcDNA synthesis was carried out using 5 mg total RNA, which wasincubated for 45 minutes in 20 mL reverse-transcriptase (RT) bufferwith 40 U RNAsin and 2mmol/L poly-dT primer and 200 units Molo-ney murine leukemia virus (MMLV) RT. The RT reaction wasstopped by heating the mixture at 95°C for 5 minutes. One tenth ofthis reaction was used as DNA template for the polymerase chainreaction (PCR) of the human IFN-a receptor, using specific primers(59-AGC GAT GAG TCT GTC GGG; 39-GGC GTG GAG CCA CTGAAC). Amplification was carried out in 10 mmol/L Tris HCl (pH 9)containing 50 mmol/L KCl, 0.01% Triton X-100, 1.5 mmol/L MgCl2,200 mmol/L dNTPs each, 50 pmol/L of each primer, and 2.5 U Ther-mus aquaticus polymerase for 30 cycles. Amplification parameterswere as follows: 30 seconds denaturation at 94°C, 60 seconds anneal-ing at 60°C, and 90 seconds extension at 72°C. An additional 10-minute extension period was added to the final cycle. Ten microlitersof the PCR products were separated by electrophoresis in a 1.2%agarose gel, which was subsequently stained in a 0.1% ethidiumbromide solution for 10 minutes. PCR products were then visualizedon a ultraviolet transilluminator. Size determination of the amplifi-cates was deduced from a DNA ladder electrophoresed in parallel.

Transactivation Assay. The ability of IFN-a to stimulate transacti-vation of an interferon-stimulated response element (ISRE) was de-termined in transient transfection assays with an ISRE-Luciferasereporter gene construct. The ISRE-luc construct, containing residues–206/286 of the human 29,59oligo-A synthetase enhancer, and amutated variant were a generous gift from Shoumo Bhattacharya(Boston, MA) and were subcloned into the pTGL2 promoter vector(Promega, Madison, WI). Transfection efficiency was monitored us-ing a b-Gal reporter construct containing the constitutive activeCMV promoter. Two 3 105 cells/well were plated in 6 well tissueculture dishes and transfected with the constructs (1.5 mg) by meansof the calcium phosphate precipitation technique using the “DNATransfection Kit” (5 Prime-3 Prime, Boulder, CO) according to man-ufacturer’s instructions. Transfection was carried out for 6 hours.Cells were allowed to recover overnight followed by stimulation for24 hours with the indicated IFN-a concentrations. Lysates of thecells were prepared, and luciferase and b-Gal activity were measuredaccording to manufacturer’s instructions (Promega, Madison, WI).

Western Analysis. Proteins were isolated in ice cold lysis buffer(150 mmol/L NaCL, 20 mmol/L Tris/HCl [pH 7.8], 2 mmol/L EDTA,50 mmol/L b-glycerophosphate, 0.5% TNP 40, 1% glycerol, 1mmol/L DTT, 10 mmol/L NaF, 10 mg/mL leupeptin, 10 mg/mL apro-tinin, 2 mmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L so-dium orthovanadate). Laemmli buffer was added, the samples wereboiled for 5 minutes, and aliquots were separated by SDS-PAGE.

HEPATOLOGY Vol. 33, No. 2, 2001 MURPHY ET AL. 347

Samples were electroblotted onto PVDF membranes and blocked inPBST containing either 5% BSA (for Cdk2 and Cdc2 antibodies ) or5% nonfat dry cow milk (all other antibodies) for 2 hours at roomtemperature. Incubation with the primary antibody was carried outovernight at 4°C. After washing in PBST, the blots were incubatedwith horseradish peroxidase-conjugated second antibody, followedby washing. Bands were visualized using the enhanced chemilumi-nescence (ECL) system from Amersham. Quantification of band in-tensity was carried out with help of the computer program ScionImage (Scion Corporation, Frederick, MD).

Determination of Phosphorylation of Stat1, Stat2, Jak1, and Tyk2 by Im-munoprecipitation Analysis. After treatment with/without 1,000 IU/mLIFN-a for the indicated times, cells were rinsed in ice cold phos-phate-buffered saline buffer and lysed in IP lysis buffer (150 mmol/LNaCL, 20 mmol/L Tris/HCl [pH 7.8], 2 mmol/L EDTA, 50 mmol/LGlycerophosphate, 0.5% TNP 40, 1% glycerol, 1 mmol/L DTT, 10mmol/L NaF, 10 mg/mL leupeptin, 10 mg/mL aprotinin, 2 mmol/Lphenylmethylsulfonyl fluoride). The lysates were sonicated, and celldebris was removed by centrifugation (10,000 rpm at 4°C for 15minutes). Aliquots of equal volume and protein content were pre-cleared for 1 hour with protein A-sepharose beads and then incu-bated overnight at 4°C with 10 mg of primary antibody (Stat1, Stat2,Jak1, or Tyk2) precoated onto protein A-sepharose beads under gen-tle rotation. Immunoprecipitates were collected, washed 4 timeswith lysis buffer and subsequently eluted by boiling in Laemmlibuffer. Tyrosin-phosphorylation of the precipitated proteins was an-alyzed by immunoblotting using an a-phosphotyrosine antibody(0,2 mg/mL). To confirm that equal amounts of immuncomplexeswere analyzed, the membranes were stripped in a solution of 62.5mmol/L Tris-HCl, pH 6.8, 2% SDS, and 100 mmol/L 2-mercaptoetha-nol for 30 minutes at 55°C and resubmitted to immunoblotting withthe antibody utilized for the immunoprecipitation.

Determination of Cdk2 and Cdc2 Activity. Activity of the cyclin de-pendent kinases was determined after immunoprecipitation. Cellswere lysed by mild sonication in ice-cold ELB buffer (50 mmol/LHEPES pH 7.5, 250 mmol/L NaCl, 5 mmol/L EDTA, 0.1% NP-40, 1mmol/L DTT, 1 mmol/L NaF, 0.1 mmol/L Na3VO4, 2mg/mL aproti-nin, 5 mg/mL leupeptin, 0.1 mmol/L PMSF) followed by a 30-minuteincubation on ice with occasional vortexing. After brief centrifuga-tion (10 min, 4°C, 13,000g) lysates (500 mg/sample) were preclearedfor 1 hour with protein A-Sepharose beads. Immuncomplexes werethen precipitated by addition of protein A-Sepharose beads pre-coated with an excess of the Cdc2 or Cdk2 antibody (10 mg). Sampleswere incubated with gentle agitation at 4°C for 4 hours. Immuncom-

plexes were subsequently washed 4 times in ice cold ELB buffer andtwice in 50 mmol/L HEPES pH 7.5 containing 1 mmol/L DTT. Kinaseactivity was determined by addition of 30 mL kinase buffer contain-ing 50 mmol/L HEPES pH 7.5, 1 mmol/L DTT, 10 mmol/L MgCl2, 1mg calf histone H1/sample, 50 mmol/L ATP, and 5 mCi of (g-32P)ATP/sample. Kinase reactions were stopped after 30 and 5 minutes forCdc2 and Cdk2, respectively, by boiling the samples in Laemmlibuffer. The reactions were separated by 10% SDS-PAGE, and kinaseactivity was determined by autoradiography of the dried gels.

Statistics. Statistical differences between control and treatmentgroups were calculated by ANOVA (Newman-Keuls) and consideredsignificant at P , .05. Values are given as mean 6 SEM unless oth-erwise stated.

RESULTS

IFN-a Signal Transduction in HCC Cell Lines. Three humanHCC cell lines were utilized throughout the study (HepG2,HuH7, and SK-Hep-1), reflecting the well-differentiated(HepG2) and the poorly differentiated (SK-Hep-1) phenotypeencountered in HCC.

Because loss of expression or function of the IFN-a recep-tor and/or components of the subsequent signaling cascademay account for the IFN-resistance encountered in individualtumors, we initially attempted to confirm functionally intactIFN-a–dependent signal transduction in the HCC cell lines.First, the expression of IFN-a receptors was determined byRT-PCR using IFN-a receptor cDNA-specific primers. Thepredicted 639 base pair (bp) amplificate was detected in eachof the HCC cell lines tested (Fig. 1).

To examine the integrity of IFN-a receptor-initiated signaltransduction, we next evaluated activation of downstream ef-fectors in the IFN-a signal transduction pathway. Accord-ingly, Jak1, Tyk2, Stat1, and Stat2 were immunoprecipitatedfrom cells incubated with IFN-a (103 IU/mL) for various timeperiods. Phosphorylation on tyrosine-residues, which reflectsthe activation state of these signaling components, was thenevaluated by immunoblotting with an antiphosphotyrosineantibody. IFN-a treatment resulted in a rapid and time-de-pendent phosphorylation of the receptor-associated kinasesJak1 and Tyk2 (Fig. 2, upper panels), with maximal phos-phorylation being observed after 15 minutes. As a conse-

FIG. 1. RT-PCR analysis ofIFN-a receptors in human HCC celllines. RT-PCR products were sepa-rated on a 2% agarose gel. The re-verse transcription reaction was car-ried out in the presence (1) andabsence (2) of reverse transcriptaseto exclude artefacts resulting fromgenomic contamination. The arrowindicates the predicted band of 639bp as deduced from the DNA ladderelectrophoresed in parallel.

348 MURPHY ET AL. HEPATOLOGY February 2001

quence, enhanced phosphorylation was detected for both the91-kd and the 84-kd splice variants of Stat1, with the strongersignal observed for the 91-kd form most likely reflecting itshigher abundance in HCC cells. An analogous, time-depen-dent phosphorylation of Stat2 was also noted in the 3 celllines.

All phosphotyrosine-blots were subsequently stripped andreprobed with the respective primary antibody to verify thatequal quantities of immuncomplexes were analyzed (Fig. 2,lower panels).

IFN-a Is Capable of Transactivating Specific DNA Elements inHCC Cell Lines. On phosphorylation, Stat1 and Stat2 formtranscriptionally active heterodimers that bind to interferon-stimulated response elements (ISREs), DNA motifs found inthe promoter region of target genes. To directly determineIFN-a dependent transactivation of ISREs in HCC cells, re-portergene studies were performed. In cells transiently trans-fected with an ISRE-luciferase reporter construct, IFN-a treat-ment induced a dose-dependent increase in luciferase activity,ranging from 3- to 5-fold in HepG2 and SK-Hep-1 to approx-imately sevenfold in HuH7 cells (Fig. 3). In contrast, cellstransfected with a mutant ISRE construct revealed no change

in the level of luciferase activity (data not shown). Thus,IFN-a induced JAK/STAT-signaling resulted in specific trans-activation of IFN regulated genes in HCC cell lines.

IFN-a Inhibits Anchorage-Dependent Growth. Having establishedan intact IFN-a signaling machinery in all three HCC cell lines, wenext evaluated the growth regulatory effects of IFN-a. Prolifera-tion of HepG2, HuH7, and SK-Hep-1 cells was determined over aperiod of 96 hours in the presence or absence of 1,000 IU/mLIFN-a. In all 3 cell lines, IFN-a-treatment resulted in a time-de-pendent reduction of cell numbers (Fig. 4), with first significanteffects observed at 72 hours in HepG2 and SK-Hep-1 cells. Growthinhibition of HuH7 was slower in onset and did not reach signifi-cance before 96 hours, which most likely reflected the comparablyslower growth rate of this cell line under control conditions. At 96hours of treatment, cell numbers had decreased to 69.7 62.5% of control in HepG2, 55.3 6 10% in HuH7 and to 52. 7 62.4% in SK-Hep-1 cells. The antiproliferative action of IFN-awas also dose-dependent (Fig. 5) with a half maximal inhibi-tion of proliferation calculated at 96 6 1.8 IU/mL for HepG2,at 90 6 1.4 IU/mL for SK-Hep-1, and at 362 6 1.5 IU/mL forHuH7 cells.

FIG. 2. Activation of the Jak-STAT signal transduction pathway in HCC cell lines. Cell lines were stimulated with IFN-a (103 IU/mL) for the indicated timeperiods and immunoprecipitations were performed with antibodies to Jak1, Tyk2, Stat1, and Stat2. Phosphorylation status of these proteins was thendetermined by Western blot analysis using the a-phosphotyrosine antibody PY-20 (upper band). Blots were subsequently stripped and reprobed with theprimary antibody (lower band) to control the amount of immunoprecipitated protein analyzed.

HEPATOLOGY Vol. 33, No. 2, 2001 MURPHY ET AL. 349

Cell Cycle Analysis of IFN-a–Treated HCC Cells Reveals S-PhaseAccumulation. To gain first insights into the mechanisms ofIFN-a–induced growth inhibition, we next analyzed cell-cy-cle distribution in response to IFN-a treatment by flow cy-tometry. Based on the earlier onset of the antiproliferativeactions of IFN-a in HepG2 and SK-Hep-1 cells, these cell lines

FIG. 4. IFN-a inhibits growth in HCC cells: time course. Subconfluentcell monolayers were treated with IFN-a (103 IU/mL) for the indicated timeperiods and cell numbers were determined. Data represent the means 6 SEMof 3 experiments carried out in triplicate. (*P , .05.)

FIG. 3. IFN-a transactivates an ISRE luciferase reporter construct. Each ofthe HCC cell lines was transfected with 1.5 mg of the ISRE luciferase reporterconstruct. After a recovery period of 24 hours, cells were treated with IFN-aat the indicated concentrations for an additional 24 hours, harvested and thenluciferase activity (RLU) was measured. The data shown represent means 6SEM of 3 experiments conducted in triplicates.

350 MURPHY ET AL. HEPATOLOGY February 2001

were chosen as representative models for a detailed analysis ofcell cycle effects. In addition, cell populations were synchro-nized in G1 to allow a more distinct representation of theirprogression through the individual cell cycle phases. Syn-chronization was achieved using serum starvation, which rou-tinely retained approximately 70% to 85% of cells in the G0/

G1-phase. Cells were then stimulated to re-enter the cell cycleby addition of fetal calf serum, and cell cycle progression wasmonitored for up to 30 hours both in the presence or absenceof IFN-a (shown in Fig. 6A for SK-Hep-1 cells). For a quan-titative evaluation of cell cycle redistribution, the percentageof cells in the G1-, S-, and G2/M-phase of IFN-a–treated cul-tures were determined and compared with the respective frac-tions in untreated time-matched controls (Fig. 6B).

Serum-stimulated synchronized SK-Hep-1 cultures exitedG0/G1 between 12 and 20 hours following addition of FCS,irrespective of IFN-a treatment (Fig. 6B, first panel). In con-trol cells, the subsequent passage through S-phase was com-pleted at 24 hours as evidenced by a decrease in the S-phasefraction (Fig. 6B, second panel, open circles) paralleled by anincreased G2/M phase population (Fig. 6B, third panel, opencircles). In contrast, S-phase progression was delayed in IFN-a–treated SK-Hep-1 cells, resulting in a significantly increasedS-phase fraction after 20 hours of IFN-a treatment (Fig. 6B,second panel, closed circles). At 30 hours, a decrement in thestill significant S-phase accumulation was noted accompaniedby an increase in the G2/M phase, suggesting that treatedSK-Hep-1 cells resumed cell-cycle progression in spite of thecontinued presence of IFN-a.

A very similar pattern of cell-cycle redistribution was ob-served in HepG2 cells (Fig. 6B). However, the time course ofcell-cycle transition appeared faster such that the G1 exit wasalmost completed at 12 hours after release from serum starva-tion. Again, IFN-a treatment resulted in a pronounced in-crease of the S-phase population.

Neither one of the HCC cell lines presented an accumula-tion of cells in the G1-phase, indicating an unhindered pas-sage of IFN-a–treated cells through the G1 checkpoint. Fur-thermore, no significant increase of cells with subdiploidDNA-content was noted in IFN-a–treated cultures (Fig 6Aand B), suggesting that the antiproliferative action did notresult from apoptosis. Taken together, the cell-cycle analysesindicated that IFN-a–induced cell growth inhibition was de-termined by S-phase regulatory factors.

IFN-a Inhibits Cdk2 and Cdc2 Kinase in SK-Hep-1 Cells. For amore detailed study on the mechanisms of IFN-a–dependentS-phase delay, SK-Hep-1 cells were chosen, because of themore persistent synchronization achieved in these cells. Dur-ing late G1, Rb phosphorylation permits derepression of E2Ffamily transcription factors, which in turn direct the tran-scription of genes required for S-phase progression, includingCyclin A and Cyclin E. Therefore, Rb expression and phos-phorylation were determined in whole cell lysates of SK-Hep-1 cells synchronized and treated as described earlier (Fig.7A). As expected, the hypophosphorylated, faster migratingform of Rb was present in starved control cultures before theaddition of serum. On serum stimulation, this faster migratingband disappeared as cells exited from G1 and, irrespective ofIFN-a treatment, was no longer detectable 12 hours after mi-togen exposure, indicating that the cytokine did not preventRb-phosphorylation in HCC cells. Similarly, the level of theG1-phase associated Cyclin E was not affected by IFN-a until20 to 24 hours after stimulation, when an increase was occa-sionally noted in treated cultures. Taken together, these re-sults are in good agreement with the FACS analyses and placethe IFN-a–dependent cell cycle regulatory events beyond thepassage of the G1 restriction point.

FIG. 5. IFN-a inhibits growth in HCC cells: dose response. Subconfluentmonolayers were treated for 96 hours with the indicated concentrations ofIFN-a. Cell numbers were then determined. Data shown is the mean 6 SEMof 3 experiments, each conducted in triplicate. (*P , .05.)

HEPATOLOGY Vol. 33, No. 2, 2001 MURPHY ET AL. 351

The subsequent progression of cells through the S-phaseand exit into G2/M are propagated by the sequential activationof Cdk2-Cyclin A, Cdc2-Cyclin A, and Cdc2-Cyclin B com-plexes, which were therefore examined next (Fig. 7). Westernanalysis of total protein lysates showed an induction in cellu-lar Cyclin A levels as control cells progressed from G1 intoS-phase. This Cyclin A induction was invariably reduced intime-matched IFN-a–treated cultures. Similarly, IFN-a treat-ment blunted the increase of cellular Cyclin B content preced-ing G2/M phase. In contrast, Cdk2 expression was not signif-icantly affected throughout the whole experiment, whereas areduction of Cdc2 expression was noted in individual exper-iments (Fig. 7A and B).

Additional control of CDK activity is exerted by inhibitorsof cyclin dependent kinases (CKIs). To examine if an induc-tion of p21cip1 or p27kip1 CKIs participates in IFN-a–mediatedgrowth inhibition in HCC cells, Western blot analyses wereperformed. As expected, the overall cellular level of p27kip1

decreased slightly whereas p21cip1 levels increased as cells re-entered the cell cycle. However, the abundance of both p21cip1

and p27kip1 were not affected by IFN-a treatment when com-pared with their levels in time-matched controls. Thus, theIFN-a–induced S-phase accumulation in HCC cells occursindependent of cellular CKI levels.

The CDC25 family of dual specificity phosphatases pro-vides a third mechanism of controlling CDK activity withCdc25A required for full activation of Cdk2 and Cdc25C forCdc2 activity. We therefore examined Cdc25 expression insynchronized mitogen-stimulated SK-Hep-1 cultures in thepresence or absence of IFN-a (Fig. 7). As expected, expres-sion of Cdc25A was regulated in a cell-cycle dependent man-ner in control cells, whereas Cdc25C levels remained un-changed. IFN-a treatment had no effect on Cdc25Aexpression, but reduced cellular Cdc25C levels at 12, 20, and24 hours. Thus, IFN-a–dependent regulation of Cdc25 ex-pression could participate in the cell cycle effects via inhibi-tion of Cdc2, but not Cdk2 activity.

To directly examine the effects of IFN-a on the activity ofCDK complexes acting at S- and G2/M-phase transitions,histone H1 kinase assays were performed on Cdk2 and Cdc2complexes from synchronized SK-Hep-1 cells. These kinaseassays revealed a marked early inhibition of Cdk2 activity inIFN-a treated cultures compared with time-matched controlcells (Fig. 8A), which persisted for up to 20 hours. Similarly,Cdc2 activity was reduced by IFN-a (Fig. 8B); however thisinhibition was first observed 20 to 24 hours after IFN-a incu-bation. Thus, IFN-a inhibited both Cdk2 and Cdc2 activity.

FIG. 6. Effects of IFN-a on cell cycle distribution of HCC cells. (Upper panels) Cell cycle progression of synchronized SK-Hep-1 populations grown in theabsence (control) or presence of IFN-a (103 IU/mL) at the indicated times after release from serum starvation (A). (Lower panels, B) A summarized time courseof IFN-a-induced cell cycle redistribution in Sk-Hep-1 and HepG2 cells (E, control conditions;F, 1,000 IU/mL IFN-a). Data shown represent the mean 6 SEMof 5 to 10 independent experiments.

352 MURPHY ET AL. HEPATOLOGY February 2001

Taken together, the observed changes in cell cycle regula-tory molecules were consistent with a significant, but tran-sient inhibition of Cdk2 activity resulting from reduced Cy-clin A levels. Subsequent cell cycle events required for theactivation of Cdc2, such as induction of Cyclin B and Cdc2 aswell as expression of Cdc25C were inhibited (Fig. 7), eithersecondary to the impairment of Cdk2 or because of indepen-dently acting mechanisms.

DISCUSSION

In view of the diverse genetic background of HCC, anygiven therapeutic approach may conceivably yield inconsis-tent and varying responses. Without the identification of rel-evant target molecules, it will be difficult to understand andinterpret the results of clinical trials in such a hetergenouspatient population. This is clearly an issue in the case ofIFN-a, which may affect multiple aspects of HCC, i.e., riskfactors, the hepatocyte transformation process, and later stepsin tumor progression via distinct and separate mechanisms.Thus, the current study undertook a detailed attempt to elu-

cidate one of these aspects, the mechanisms of direct antipro-liferative actions of IFN-a in HCC cells.

Initially, the expression of functionally intact IFN-a recep-tors was established, and the activation of STAT transcriptionfactors was confirmed. Stat1 has been critically implicated inthe growth effects of type I interferons in studies utilizingStat1-deficient cells34 and targeted gene disruption in trans-genic mice.35 Furthermore, molecular defects in the STATactivation pathway are observed in some human tumors.36

However, all 3 cell lines readily showed Stat1 phosphorylationand ISGF3 transactivation on IFN-a treatment, suggestingthat IFN-a–dependent signal transduction is retained inHCC.

With respect to the most common genetic lesions impli-cated in HCC, i.e., loss of the tumor suppressors p16ink47,37

and p53,8 the panel of cell lines that we used reflected some ofthe heterogeneity in the genetic background: HepG2 cells ex-press functionally intact p16ink438 and p53.9,39 Conversely, SK-Hep-1 are p16ink4 deficient (data not shown) but possess func-

FIG. 7. Effects of IFN-a on cell cycle regulatory proteins in SK-Hep-1 cells. (A) Whole cell lysates were isolated from control and IFN-a–treatedsynchronized SK-Hep-1 cells after the indicated time and subjected to Western analysis. (B) Band intensity was determined by densitometry in 2 to 3independent experiments. Values obtained in IFN-a–treated cell populations after 24 hours of culture with 103 IU/mL IFN-a are expressed as a percentage ofthe values in time-matched controls.

HEPATOLOGY Vol. 33, No. 2, 2001 MURPHY ET AL. 353

tional p53 (in spite of a rearrangement in one allele).9,39

Finally, HuH7 cells lack both functional p16ink440 and p53.9,39

Despite their genetic heterogeneity, IFN-a treatment uni-formly inhibited proliferation of all 3 HCC cell lines. Al-though cell doubling times were significantly prolonged,IFN-a did not completely block proliferation as cells contin-ued to grow even at maximal concentrations of IFN-a. Inagreement with these observations, cell cycle analyses per-formed in synchronized HCC-cell populations did not reveal adistinct cell cycle block, but rather a prolongation of the timeperiod required to progress through S-phase. In unsynchro-nized cell populations, this corresponded to a distinct in-crease in the S-phase population (data not shown), and thusreproduced the IFN-a–dependent cell cycle redistributionpreviously reported by Yano et al.,29 who recorded an in-creased S-phase fraction in 10 out of 11 human HCC cell lines.However, in contrast to their results, we could not observeapoptosis in IFN-a–treated HCC cell cultures. BecauseHepG2 and SK-Hep-1 cells readily undergo apoptosis in re-sponse to chemotherapeutic drugs such as 5-fluorouracil orcisplatin,41 this discrepancy does not reflect an apoptosis-re-sistant phenotype of the cell lines utilized in the current study.Also, both studies obtained comparable EC50s for the growthinhibitory effects of IFN-a, excluding relevant differences inthe sensitivity of the cell lines to the cytokine. Furthermore,the failure of IFN-a to induce apoptosis was not the result ofp53 deficiency, as HepG2 and SK-Hep-1 cells analyzed in thecurrent study express wild-type p53 and, inversely, the HAK1cells reported to undergo apoptosis in response to IFN-a carrya p53 mutation.42

Irrespective of these differences, both studies consistentlyidentified an IFN-a–induced S-phase accumulation as theprevalent cell cycle alteration, suggesting that the molecularmechanism of IFN-a action is highly conserved between dif-ferent HCC tumor collectives. This is noteworthy becauseIFN-a–dependent cell cycle effects in IFN-a–sensitive leuke-

mia and lymphoma cell lines43-46 are directed primarily at theG1/S transition and suggests that IFN-a differentially targetsspecific cell cycle components in different tumor entities. InG1-arrest models, the cell cycle effects are generally Rb-depen-dent and mediated by an increase in the cellular comple-ment of CKIs, such as p15,47 p19,44,49 p21cip1,47,49,50 andp27kip1.47,51,52

Based on the IFN-a–induced cell cycle redistribution ob-served in the current study, neither p15 nor p16ink4 were in-vestigated because these CKIs do not inhibit kinases involvedin later cell cycle phases.53 However, regulation of the lessrestricted CDK inhibitors p21cip1 and/or p27kip154 was exam-ined in SK-Hep-1 cells. Upregulation of both CKIs by gin-sengoid compounds has previously been reported to result ina G1-phase arrest in SK-Hep-1 cells,55,56 indicating that theCDK inhibitors are functionally competent in HCC cells whenadequately stimulated. In the current study, p21cip1 and p27kip1

were found to be regulated cell cycle dependent but not inresponse to IFN-a treatment.

Although most studies on IFN-a–induced cell cycle regu-lation have observed inhibitory effects on G1 to S-phase tran-sition, other studies revealed S-phase accumulation in re-sponse to IFN-a treatment in vivo57 and in vitro.58-61 S-phaseregulatory pathways were proposed to prevail under condi-tions of a severely disrupted Rb-dependent G1 checkpointcontrol, as is often encountered in epithelial tumors.28 How-ever, the mechanisms that operate in the transformed cellmodels to cause S-phase accumulation have remained elusive.Our data on cell cycle distribution show that IFN-a treatmentprevented neither G1-exit nor entry into the S-phase of thecell cycle in synchronized HCC cultures. In sharp contrast,induction of Cyclin A, which occurred in the synchronizedcontrol populations as the cells traversed the restriction point,was distinctly reduced by IFN-a stimulation. Because CyclinA expression is absolutely required for passage through S-phase,62 the decrease of cellular Cyclin A content represents a

FIG. 8. Effects of IFN-a on the activity of the cyclin dependent kinases Cdk2 and Cdc2 in SK-Hep-1 cells. The activity of Cdk2 (A) and Cdc2 (B) wasdetermined after the indicated times using Histone-H1 kinase assays. The upper panel shows a representative autoradiograph for Cdk2 (A) and Cdc2 (B) kinaseassays and the bar graph in the lower half of each figure summarizes the results from 3 independent experiments with the mean 6 SEM of kinase activity aftertreatment with IFN-a expressed as percentage of time-matched controls.

354 MURPHY ET AL. HEPATOLOGY February 2001

likely cause for IFN-a–dependent growth inhibition in HCC.Supporting a critical role of Cyclin A repression for IFN-amediated cell cycle inhibition, a selective down-regulation ofCyclin A in HCC cell lines similarly resulted in S-phase pro-longation or S-phase arrest.63 The capacity of IFN-a to inter-fere with Cyclin A expression in HCC is particularly notewor-thy in view of the reported overexpression of Cyclin A inapproximately 40% of tumor tissues from HCC-patients.64

Furthermore, an HBV integration into the human Cyclin Agene has been documented in an early HCC that resulted inthe production of a chimeric, degradation-resistant Cyclin Aprotein, responsible for enhanced expression of Cyclin A inthe tumor tissue.65,66

As a consequence of Cyclin A inhibition, subsequent cellcycle events were impaired in IFN-a–treated HCC cell popu-lations, as evidenced by significant downregulation of CyclinB. Cyclin B is synthesized to function as a regulatory subunitin Cdc2 kinase complexes as cells progress from S- into G2/M,67 and the peak of cellular Cyclin B levels observed at theG2/M transition is largely dependent on Cdk2 activity.68

Thus, the lack of Cyclin B induction observed in the IFN-a–treated HCC cells likely reflects the inhibition of Cdk2 activ-ity. Similarly, the cellular Cdc2 content, which analogous toCyclin B depends on Cdk2/E2F mediated induction,69 ap-peared reduced under these conditions but this effect did notreach statistical significance. The comparatively strong inhi-bition of Cyclin B expression might therefore be driven byadditional mechanisms specifically affecting this cyclin. Sucha Cdk-2–independent, IFN-a–induced inhibition of Cyclin Bexpression also appears to operate in human neuroendocrinetumor cells.70

Although reduced Cyclin B levels in IFN-a–treated SK-Hep-1 cells could account for an inhibition of Cdc2 kinaseactivity, additional downregulation of Cdc25C phosphatasemight well contribute to the inhibitory mechanism. Cdc25Cphosphatase dephosphorylates Cdc2 on both Thr-14 andTyr-15 in late G2, leading to the activation of Cdc2-Cyclin Bcomplexes.71,72 As Cdc25C expression is thought to be cellcycle independent,73,74 the reduction of Cdc25C cannot beexplained as a mere consequence of reduced Cdk2 activity,but would represent a second, independent pathway of cellcycle inhibition at the G2/M transition. Despite the fact thatthis mechanism was not rate limiting in SK-Hep-1 cells al-ready arrested in the S-phase of the cell cycle, it might operatein cells that arrest in the G2/M phase in response to type 1interferons.

In summary, the current study provides for the first time adetailed analysis of the direct, cell cycle regulatory effects ofIFN-a in HCC cells. We show a direct antiproliferative actionof IFN-a that results in a substantial but transient delay inS-phase progression. We have identified Cyclin A, Cyclin B,and Cdc25C as relevant, cell cycle regulatory target moleculesof IFN-a in HCC. Via down-regulation of these targets, IFN-ais capable of modulating 2 independent cell cycle controlpathways affecting either the S-phase or the G2/M transition.

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