Cell cycle deregulation in liver lesions of rats with and without genetic predisposition to hepatocarcinogenesis

Cell Cycle Deregulation in Liver Lesions of Rats Withand Without Genetic Predisposition to

HepatocarcinogenesisRosa M. Pascale, Maria M. Simile, Maria R. De Miglio, Maria R. Muroni, Diego F. Calvisi, Giuseppina Asara,

Daniela Casabona, Maddalena Frau, Maria A. Seddaiu, and Francesco Feo

Preneoplastic and neoplastic hepatocytes undergo c-Myc up-regulation and overgrowth inrats genetically susceptible to hepatocarcinogenesis, but not in resistant rats. Because c-Mycregulates the pRb-E2F pathway, we evaluated cell cycle gene expression in neoplastic nodulesand hepatocellular carcinomas (HCCs), induced by initiation/selection (IS) protocols 40and 70 weeks after diethylnitrosamine treatment, in susceptible Fisher 344 (F344) rats, andresistant Wistar and Brown Norway (BN) rats. No interstrain differences in gene expressionoccurred in normal liver. Overexpression of c-myc, Cyclins D1, E, and A, and E2F1 genes, atmessenger RNA (mRNA) and protein levels, rise in Cyclin D1-CDK4, Cyclin E-CDK2, andE2F1-DP1 complexes, and pRb hyperphosphorylation occurred in nodules and HCCs ofF344 rats. Expression of Cdk4, Cdk2, p16 INK4A, and p27 KIP1 did not change. In nodulesand/or HCCs of Wistar and BN rats, low or no increases in c-myc, Cyclins D1, E, and A, andE2F1 expression, and Cyclin-CDKs complex formation were associated with p16 INK4A over-expression and pRb hypophosphorylation. In conclusion, these results suggest deregulationof G1 and S phases in liver lesions of susceptible rats and block of G1-S transition in lesionsof resistant strains, which explains their low progression capacity. (HEPATOLOGY 2002;35:1341-1350.)

Previous studies on genetic predisposition to hepa-tocellular carcinoma (HCC) of rats led to identifi-cation of 4 hepatocarcinogenesis susceptibility

(Hcs1-4) loci, and 7 resistance (Hcr1-7) loci1,2 (De Miglioet al., unpublished data). Resistance alleles, dominantlytransmitted to the progeny,3,4 apparently modify the ac-tivity of susceptibility loci. Recent evidence suggests thepresence of at least 3 oncosuppressor genes at Hcr1 locus.4

Molecular mechanisms underlying these effects are un-

known. Available evidence suggests the existence of a rel-atively stable genome in neoplastic lesions of resistant rats,as shown by the absence of c-myc amplification in thelesions of a resistant Wistar rat strain, which instead ispresent in susceptible Fisher 344 (F344) rats.5,6 More-over, allelic imbalance occurs, at several chromosomes, inHCCs of susceptible (F344 x Wistar Furth)F1 rats,7 butnot in those of resistant BFF1 rats.8 HCCs induced inLFF1 rats, generated by crossing the susceptible F344 andLong-Evans strains, show allelic imbalance at Hcs1, Hcr1,and Hcr6.4 c-myc is located at Hcs1 in a segment syntenicto human chromosomal regions in which frequent allelicgain occurs.9 These observations suggest that mechanismscontrolling cell growth are differently affected in neoplas-tic liver lesions of susceptible and resistant rats.

Overexpression of c-myc in c-myc– and c-myc/Tgf-�–transgenic mice is associated with deregulation of thepRb-E2F pathway.10 Interaction of c-Myc with variouscell cycle components occurs in in vitro growing cells.11,12

Enzymes controlling cell cycle include cyclin-dependentkinases (CDKs), activated by binding to the cyclins.13

Complexes of CDK4 and CDK6 with D-type cyclins arerequired for the G1 phase progression. Further progres-sion through G1 requires cyclin E, and passage through

Abbreviations: HCC, hepatocellular carcinoma; CDK, cyclin-dependent kinase;BN, Brown Norway; AAF, 2-acetylaminofluorene; GST-P, glutathione-S-trans-ferase; IP, intraperitoneal; RT-PCR, reverse transcriptase polymerase chain reac-tion; F344, Fisher 344; IS, initiation/selection; ISS, initiation/selection/selection;SD, standard deviation; TK test, Tukey-Kramer test; TGF-�, transforming growthfactor �.

From the Department of Biomedical Sciences, Division of Experimental Pathol-ogy and Oncology, University of Sassari, Sassari, Italy.

Supported by funds from the Italian Association for Cancer Research (AIRC), theScientific Research Ministry (MIUR), and the Health Department of the RegionalSardinian Government (RAS).

Address reprint requests to: Francesco Feo, M.D., Dipartimento di Scienze Bio-mediche. Sezione di Patologia Sperimentale e Oncologia. Universita di Sassari, ViaP. Manzella 4, 07100 Sassari, Italy. E-mail: [email protected]; fax: (39)079-228485.

Copyright © 2002 by the American Association for the Study of Liver Diseases.0270-9139/02/3506-0008$35.00/0doi:10.1053/jhep.2002.33682

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the S phase requires cyclin A, each in the complex withCDK2. CDK activity may be curtailed by various inhib-itors including p16 INK4A, p27 KIP1, and p21WAF1. G1 cyclins(D and E types), in complex with CDKs, phosphorylateretinoblastoma protein (pRb).13 HypophosphorylatedpRb complexes E2F family transcriptor factors, thus halt-ing cell cycle. After pRb phosphorylation, E2F formscomplexes with DP1, thus it acts on DNA synthesisgenes.14,15 Overexpression of c-MYC, CYCLIN D1,CYCLIN A, and CYCLIN E,9,16-18 and underexpression ofp16 INK4a and p27 KIP1 genes19,20 occur in human HCCs,but the role of these alterations in early liver lesions andtheir relationships with genetic susceptibility to HCC areunknown.

Chemically induced neoplastic nodules in rat livershow several morphologic, biochemical, and molecularcommonalties with neoplastic and dysplastic nodules pre-ceding the development of human HCC.9 The rat modelof hepatocarcinogenesis allows detailed analysis of molec-ular mechanisms involved in genetic predisposition, inrelatively early stages of hepatocarcinogenesis. In the at-tempt to relate cell cycle deregulation to genetic predis-position to liver cancer, this report evaluates therelationships between the phenotypic behavior and theexpression of various positive and negative signals impli-cated in the progression of G1 and S phases of the cellcycle in neoplastic nodules and HCCs induced in ratstrains with different susceptibility to hepatocarcinogen-esis.

Materials and Methods

Animals and Treatments. Male F344, Wistar, andBrown Norway (BN) rats (Charles River Italia, Calco,Italy; 160-180 g body weight) were housed individually insuspended wire-bottomed cages in a room with constanttemperature (22°C) and humidity (55%) and with a 12-hour light (6:00 A.M.-6:00 P.M.) and dark (6:00 P.M.-6:00A.M.) cycle. Five untreated rats of each strain were used asa source of normal liver. The other rats were initiated witha single intraperitoneal (IP) dose (150 mg/kg) of dieth-ylnitrosamine, and subjected, 2 weeks later, to a 15-dayfeeding of a diet (Type 52; Piccioni, Gessate, Milano,Italy) containing 0.02% 2-acetylaminofluorene (AAF),with a partial hepatectomy at the midpoint of this treat-ment (initiation-selection [IS] protocol).21 Wistar rats arerelatively resistant to AAF, and IS protocol induces veryfew, small HCCs5 not suitable for molecular analyses. Toenhance tumorigenesis, a subgroup of Wistar rats wassubjected, 2 weeks after the first AAF treatment, to asecond AAF treatment in which a single CCl4 administra-tion (0.2 mL/100 g 1:2 in olive oil, by gavage) substituted

partial hepatectomy (initiation/selection/selection [ISS]protocol).5 Animals were killed at 40 and 70 weeks bybleeding through the thoracic aorta under ether anesthe-sia to collect neoplastic nodules and HCCs, respectively.All animals received humane care, and the study protocolswere in compliance with our institution’s guidelines foruse of laboratory animals.

Tissue Collection and Histology. Forty weeks afterinitiation, livers from F344 and Wistar rats were resectedand cut into 2- to 3-mm slices and nodules with diametersof 3 mm or greater were collected, leaving, whenever pos-sible, a thin rim of nodular tissue around each lesion toavoid contamination by non-neoplastic parenchyma.Very small nodules present at this time in BN rats werenot collected. At 70 weeks, large nodules were collectedfrom all rat strains. Small pieces of each nodule and HCCwere fixed in neutral, buffered paraphormaldehyde, em-bedded in paraffin, sectioned into 5-�m thick slices, andused for hematoxylin-eosin staining or glutathione-S-transferase, placental form (GST-P) immunohistochem-istry.4 The remaining portion of the lesions were frozen inliquid nitrogen and kept at �80°C until used for furtheranalyses. The number of microscopic lesions per cubiccentimeter of liver and the mean volume of lesions weredetermined by computer-assisted morphometric analy-sis.22 To determine pulse-labeling index, the rats receivedan IP injection of 5 mg/100 g body weight of 2-bromo-3�-deoxyuridine, 2 hours before killing. 2-Bromo-3�-de-oxyuridine incorporation into nuclei was determinedimmunohistochemically by the cell proliferation kit (Am-ersham Pharmacia Biotech, Cologno Monzese, Milan, It-aly). Remodeling lesions were identified as areas lackinguniformity of GST-P immunostaining.23 Neoplasticnodules and HCCs were classified on the basis of thepublished criteria.24

Comparative RT-PCR. Semiquantitative reverse tran-scriptase polymerase chain reaction (RT-PCR) was per-formed as published.5,25 Briefly, 18-�L aliquots of reac-tion mixture (Titan One Tube RT-PCR System;Boehringer, Mannheim, Germany), containing the ap-propriate primers (20 pmol each; Table 1) and dNTPs(200 �mol/L each, including 5 �Ci of [�-32P]dCTP)were added to a series of 3 tubes per tissue, followed by2-�L aliquots of 3 appropriate dilutions of total RNAmaster solutions (4 �g/mL). Calibration of messengerRNA (mRNA)/complementary DNA concentration wasmade for each tissue, with primer pair specific forGAPDH reference gene. After correction of RNAamounts used, to equalize signal intensities in the differ-ent tissue samples, a second calibration was made, if nec-essary, followed by enzymatic amplification, in thepresence of specific primers. Cycling parameters for RT-

1342 PASCALE ET AL. HEPATOLOGY, June 2002

PCR analyses were 30 minutes at 55°C, followed by 2minutes at 95°C, 1 minute at 55°C, and 1 minute at 72°Cfor 30 cycles in a Stratagene RoboCycler (Packard Instru-ments Co., Meriden, CT). PCR products were applied toa 6% polyacrylamide gel containing 10% glycerol, andInstant Imager (Packard Instruments Co.) analysis of thegels revealed single bands for all genes tested, which werequantitated on the basis of the radioactive counts on thegels dried onto 3-mm paper and were reproduced by im-age analysis software on a personal computer.

Immunoblotting. Control rat liver, pool of nodules,and HCCs were homogenized in 5 volumes of lysis buffercontaining 30 mmol/L Tris-Cl (pH 7.5), 150 mmol/LNaCl, 1% NP40, 0.5% Na deoxycholate, 0.1% sodiumdodecyl sulfate (SDS), 10% glycerol, 5 mmol/L ethyl-enediaminetetraacetic acid (EDTA), 1 mmol/L Na3VO4,20 mmol/L Na pyrophosphate, 1 mmol/L phenylmeth-ylsulfonyl fluoride, 10 �g/mL aprotinin, and 10 �g/mLof leupeptin. After 30 minutes in ice, homogenates weresonicated and centrifuged to eliminate cell debris. Super-natants were precleared with 50 �L/mL of Gamma BindG Sepharose beads (15 minutes at 4°C in lysis buffer),centrifuged and incubated for 30 minutes at 4°C afteraddition of 0.25 �g/mL of appropriate control immuno-globulin G and 20 �L/mL of protein G-agarose. Aftercentrifugation, immunoprecipitation was performed byincubating supernatants (1-2 mg protein) overnight at4°C with 4 �g of agarose-conjugated antibodies/mg ofsupernatant protein (Santa Cruz, D.B.A. Italia, Segrate,Italy; Table 2). Immunocomplexes were absorbed onGamma Bind G Sepharose beads, and washed 5-fold withHepes NaCl Triton glycerol (HNTG) buffer (20 mmol/L

HEPES, pH 7.5, 150 mmol/L NaCl, 0.1% TritonX-100, and 10% glycerol). The pellets were resuspendedin sample buffer (100 mmol/L Tris-Cl, pH 6.8, 5% SDS,5% glycerol, 0.005% bromophenol blue, and 5% 2�-mercaptoethanol), boiled for 5 minutes and centrifugedto separate immunoprecipitated protein. For negativecontrols, primary antibodies were incubated before im-munoprecipitation, with the respective immunogenpeptide (1:20 wt/wt). A total of 30 �L of immunopre-cipitated samples were separated by 10% SDS-PAGE,and transferred onto polyvinylidine difluoride (PVDF)membranes (Millipore). The membranes were washedaccording to the manufacturer’s instructions. Nonspe-cific binding was blocked by incubating for 40 minutesat room temperature with Blocking Reagent (Boehr-inger Mannheim, Roche Diagnostics S.p.A., Monza,Italy). The membranes were incubated for 1 hour at23°C with biotinylated secondary antibody (VectorLaboratories, D.B.A., Segeate, Milano, Italy) and di-luted 1:1,000. After incubation with Streptavidin-horseradish peroxidase conjugate and washing,immune complexes were visualized and quantified byenhanced chemiluminescence by using Image Master(Amersham Pharmacia Biotech). Reaction specificitywas tested by immunoblot analysis of control proteins(blocking peptides, 0.4 �g; Santa Cruz) with the cor-responding primary antibodies.

Western Blot Analysis. To quantitate Cyclin D1/CDK4, Cyclin E/CDK2, and E2F1/DP1 complexes, 40�L of the immunoprecipitates with Cyclin D1, Cyclin E,and E2F1 primary antibodies (Santa Cruz, Table 2), wereseparated by SDS-PAGE and transferred onto PVDFmembranes as described above. The blots were probedwith 1.8 �g/mL of polyclonal antibodies against CDK4,CDK2, or DP1 and, after washing, were incubated withsecondary antibodies. Immune complexes were visualizedby enhanced chemiluminescence; pRb phosphorylation

Table 2. Primary Antibodies Used for ImmunoprecipitationExperiments

Protein Antibody Epitope Mapping

c-myc Mouse monoclonal COOH terminusCyclin D1 Rabbit polyclonal NH2 terminusCyclin E Mouse monoclonal COOH terminusCyclin A Goat polyclonal COOH terminusCDK4 Goat polyclonal COOH terminusCDK2 Rabbit polyclonal COOH terminusE2F1 Mouse monoclonal Rb binding domain*pRb Mouse monoclonal COOH terminusDP1 Rabbit polyclonal NH2 terminusp16INK4 Rabbit polyclonal Full lengthp27KIP1 Mouse monoclonal Full length

*Reacting with both phosphorylated and not phosphorylated forms.

Table 1. Primers Used for RT-PCR Experiments

Gene Primers Base Pairs

c-myc Fw 5�-CTCGGAAGGACTATCCTGCTGCCAA 150Rev 5�-GGCGCTCCAAGACGTTGTGTTTCG

Cyclin D1 Fw 5�-GCCATGCTTAAGACTGAGGAGACCT 300Rev 5�-TTGCAGCAACTCCTCGGGGCGGATA

Cyclin E Fw 5�-CTGTCAGCTGACAGTGGAGAAGG 150Rev 5�-AGGGTGCTACTTGACCCACTGGA

Cyclin A Fw 5�-AACGATGAGCACGTCCCTACTGT 280Rev 5�-CAAGGATGGCCCGCATACTGTTA

E2F1 Fw 5�-TGCAGATTCTTGGGCACCTAGAG 250Rev 5�-CCAGAAGAGACCTGGCTTAAGGCTG

CDK4 Fw 5�-CAAGCGAATCTCTGCCTTTCGAGC 90Rev 5�-GGGAACATACCCCTTAGTGTAGAG

CDK2 Fw 5�-AATCCGGCTCGACACTGAGACTG 290Rev 5�-CACGGTGAGAATGGCAGAATGCTAGGCCCT

p16INK4A Fw 5�-GTCAAAGTGGCAGCTCTCCTGCT 240Rev 5�-TGTCGGTGACCCGGGAAACGTTC

p27KIP1 Fw 5�-AGCCAGCGCAAGTGGAATTTCGACT 300Rev 5�-GAAGAATCTTCTGCCGCAGGTC

Gapdh Fw 5�-GTATGACTCTACCCACGGCAAGTTC 190Rev 5�-AGCCTTCTCCATGGTGGTGAAGAC

HEPATOLOGY, Vol. 35, No. 6, 2002 PASCALE ET AL. 1343

was evaluated by separating 160 �g of total protein lysateby 10% SDS-PAGE. Proteins were then transferred ontoPVDF membranes, and reacted with 1.8 �g of anti-Rbantibody.

CDK Assay. Kinase assays were performed as de-scribed.26 Briefly, 10% homogenates of different tissueswere made in a buffer containing 50 mmol/L HEPES(pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 2.5mmol/L EGTA, 1 mmol/L DTT, 10% glycerol, 144�mol/L AEBSF, 10 �g/mL leupeptin, 10 �g/mL aproti-nin, 10 mmol/L �-glycerophosphate, 1 mmol/L NaF,and 0.1 mmol/L Na orthovanadate. Tween 20 was added(final concentration 0.1%), and after centrifugation 5 mgof liver homogenate proteins were immunoprecipitatedfor 2 hours at 4°C with protein G-sepharose beads andappropriate antibody and washed 4-fold with immuno-precipitation buffer and twice with 50 mmol/L HEPES(pH 7.5) containing 1 mmol/L DTT. Immunoprecipi-tated proteins were suspended in 30 �L of kinase buffer(50 mmol/L HEPES, pH 7.5, 10 mmol/L MgCl2, 1mmol/L DTT) containing 1 �g of GST-pRb fusion pro-tein (Santa Cruz Biotechnology), 2.5 mmol/L EGTA, 10mmol/L �-glycerophosphate, 0.1 mmol/L Na orthovana-date, 1 mmol/L NaF, 20 �mol/L adenosine triphosphate(ATP), and 10 �Ci [�-32P]ATP (Amersham PharmaciaBiotech). After 30 minutes of incubation at 30°C, thesamples were boiled in SDS-PAGE buffer and separatedby PAGE. Phosphorylated proteins were quantitated byInstant Imager.

Statistical Analysis. The values in the Figures aremeans � SD. Data regarding lesion number and volume,labeling index, remodeling, protein and mRNA levels,and CDK4 activity were analyzed by ANOVA. Multiple

comparisons were performed by the Tukey-Kramer (TK)test, and differences in tumor incidence were analyzed bythe �2 test, using GraphPad InStat 3 (www.graphpad.com). The level of statistical significance was set at P � .05.

Results

Development of Neoplastic Lesions. Forty weeks af-ter initiation, the nodule number was about 8-fold lowerin Wistar rats and slightly but significantly (P � .001)higher in BN rats than in F344 rats subjected to the ISprotocol (Table 3). Lesion size was about 5- to 7-foldlower in Wistar and BN than in F344 rats. These param-eters increased in Wistar rats subjected to ISS, but lesionnumber and volume remained 1.6-fold and 2-fold lowerthan in F344 rats subjected to IS protocol. Labeling indexchanged in different rat strains concurrently with nodulevolume, and remodeling was significantly higher in nod-ules of Wistar and BN rats (P � .001).

Nodules of F344 rats were well differentiated carcino-mas or adenomas. In Wistar and BN rats subjected to ISmost nodules constituted by clear/eosinophilic cells didnot exhibit atypical features. The number of atypical le-sions slightly increased in Wistar rats subjected to ISS, butthe percentage of well-differentiated carcinomas/adeno-mas was not higher than 26%.

Low progression capacity of nodules of Wistar and BNrats was confirmed by low incidence and multiplicity ofHCCs in these rats (Table 4). Poorly differentiated carci-nomas were present only in F344 rats, in about 71% ofwhich moderately differentiated carcinomas were seen.Of HCCs, 92% to 100% induced by IS protocol inWistar and BN rats were well-differentiated carcinomas.This figure was 75% in Wistar rats subjected to ISS.

Table 3. Development of Neoplastic Nodules in Rat StrainsWith Different Genetic Predisposition to

Hepatocarcinogenesis

F344 Wistar BN

IS IS ISS IS

Number/cm3

(�10�3) 115.0 � 12.2 14.6 � 2.6 73.3 � 6.3 135.4 � 11.7Volume

(cm3 � 104) 6.22 � 0.80 1.33 � 0.71 3.42 � 0.55 0.83 � 0.02Labeling index (%) 6.42 � 1.21 0.83 � 0.61 2.88 � 0.84 0.91 � 0.05Remodeling (%) 9.6 � 1.3 52.3 � 9.4 23.1 � 2.4 22.6 � 3.6

NOTE. Liver nodules were induced by diethylnitrosamine followed by a selectiontreatment (IS protocol) and were collected 40 weeks after initiation. Whenindicated, the rats were subjected to 2 selection treatments (ISS protocol).Labeling index is expressed as the percentage of GST-P–positive hepatocytes thatincorporated 2-bromo-3�-deoxyuridine, and remodeling is expressed as the per-centage of lesions with nonuniform GST-P immunostaining on total GST-P–positivelesions. Data are means � SD of 10 rats per each strain. TK test, F344 vs. Wistar(IS and ISS) and BN, P � .001 for all parameters tested.

Table 4. Multiplicity and Incidence of HCCs in Rat StrainsWith Different Genetic Predisposition to

Hepatocarcinogenesis

F344 Wistar BN

IS IS ISS IS

Incidence (%)* 18 (90) 3 (15) 9 (37.5) 10 (41.6)Multiplicity† 2.3 � 1.15 1.3 � 0.57 1.3 � 0.50 1.2 � 0.42Well differentiated (%) 6 (14.6) 4 (100) 9 (75) 11 (92)Moderately

differentiated (%) 29 (70.7) — 3 (25) 1 (8)Poorly differentiated (%) 6 (14.6) — — —

NOTE. Hepatocellular carcinomas were induced by diethylnitrosamine followedby a selection treatment (initiation/selection, IS, protocol) and were collected 70weeks after initiation. When indicated, the rats were subjected to 2 selectiontreatments (ISS protocol).

*The number of rats analyzed was 20 for F344 and Wistar IS, and 24 for WistarISS and BN.

†�2 test, P � .0174.


pRb Phosphorylation. pRb phosphorylation wascomparatively evaluated in livers and HCCs of suscepti-ble and resistant rat strains by immunoblotting with anantibody recognizing both the hypophosphorylated andhyperphosphorylated forms of pRb. Apparently, pRb didnot undergo significant changes in expression in HCCs ofall strains tested, but it was much more phosphorylated inHCCs from F344 rats with respect to control liver (Fig.1). Relatively low pRb phosphorylation occurred inHCCs of Wistar and BN rats. This behavior was con-firmed in all carcinomas of F344 and BN rats (n � 6) andWistar rats (n � 5) (Fig. 1).

Expression of Cell Cycle–Related Genes. c-myc, Cy-clin D1, and Cyclin E mRNA levels were 4- to 10-foldhigher in nodules and HCCs than in normal livers of

F344 rats (Fig. 2). Lower increases in Cyclin A and E2F1mRNA occurred. Very low increases in c-myc, Cyclin D1,and E2F1 mRNA and no changes of Cyclin A and CyclinE mRNA were found in nodules and HCCs induced byISS in Wistar rats and in HCCs of BN rats. No significantinterstrain differences of gene expression occurred in nor-mal liver. Consequently, mRNA levels of all genes testedwere significantly higher in nodules and HCCs of suscep-tible F344 rats than in the lesions of resistant rats (Fig. 2).

These results were confirmed by immunoprecipitationexperiments (Fig. 3) that showed marked increases in c-Myc, Cyclins D1, E, and A, and E2F1 in nodules andHCCs of F344 rats. In contrast, no increase (Cyclin E andA) or a small increase (c-Myc, Cyclin D1, and E2F1) incell cycle–related proteins occurred in nodules of Wistarrats and in HCCs of Wistar and BN rats. These resultsindicate a correlation between nodule and HCC progres-sion and overexpression of cell cycle proteins. This wasfurther shown by the complete absence of increase in ex-pression of cell cycle proteins in nodules of Wistar ratssubjected to IS protocol that do not progress to HCC (notshown) (Fig. 3).

Levels of Cyclin-CDK and E2F1-DP1 Complexesand CDK4 Activity. Evaluation of the expression ofCDK4 and CDK2 did not reveal any change at both

Fig. 2. RT-PCR from mRNA of normal liver (C), neoplastic nodules (N), and HCCs (H) of F344, Wistar, and BN rats. N and H were induced by ISprotocol, in F344 and BN rats, and by ISS protocol in Wistar rats, as described in the Materials and Methods. RT-PCR products were separated byelectrophoresis into 6% polyacrylamide gel/10% glycerol. The size of PCR products was evaluated by comparing ethidium bromide–stained RT-PCRproducts with the fragment size marker after electrophoresis in 2% agarose gel. Left: Representative reproduction by Instant Imager software ofRT-PCR–radiolabeled products. Right: Quantitative Instant Imager analysis showing mean radioactive counts � SD of 5-7 rats, normalized to Gapdhvalues (relative amounts). TK test, F344: C vs. N and H, at least P � .01 for all genes. Wistar: C vs. N and H, and BN: C vs. N, at least P � .05for c-myc, Cyclin D1, and E2F1; not significant for Cyclin E and Cyclin A. F344 vs. Wistar and BN: N and H, P � .001 and C, not significant for allgenes.

Fig. 1. Representative Western blot showing pRb hyperphosphoryla-tion in HCCs of the susceptible F344 rats but not in HCCs of Wistar andBN resistant rats. (C), normal liver; (H), HCC.


mRNA and protein levels in nodules and HCCs withrespect to normal liver in any rat strain (data not shown).Moreover, no interstrain difference in normal liver ex-pression of CDK4 and CDK2 were seen; however, com-parative evaluation of the presence of Cyclin D1-CDK4and Cyclin E-CDK2 complexes in F344 and Wistar rats(Fig. 4) showed the absence of significant interstrain dif-ferences in normal liver and 4- to 8-fold increases in nod-ules and HCCs of F344 rats, with respect to normal liver,and very small and insignificant increases in the lesions ofWistar rats (Fig. 4).

The determination of CDK4 activity (Fig. 5) revealedabout 1.9-fold increases in pRb phosphorylation in nod-ules and HCCs of F344 rats, whereas no significant in-crease occurred in the same lesions of Wistar and BN rats.These results are consistent with pRb hyperphosphoryla-tion in nodules and HCCs of F344 rats (Fig. 1), resultingin increased availability of free E2F1 and formation of theE2F1-DP1 complex, which was indeed found in nodulesand HCCs of F344 rats but not in those of Wistar rats(Fig. 4).

Up-regulation of p16INK4A in Liver Lesions of Re-sistant Rats. No changes in p16INK4A and p27KIP1 geneexpression were found, at mRNA and protein levels, innodules and HCCs of F344 rats, with respect to normalliver (Fig. 6). No interstrain differences in the expressionof these 2 genes were observed in normal liver, but 2- to

Fig. 3. Detection by immunoprecipitation of c-Myc, Cyclins D1, E, and A, and E2F1 in homogenates from normal liver (C), neoplastic nodules (N),and HCCs (H) of F344, Wistar, and BN rat strains. N and H were induced by IS protocol, in F344 and BN rats and by ISS protocol in Wistar rats,as described in the Materials and Methods. Sample proteins and blocking peptides were coimmunoprecipitated by antibodies against Myc, CyclinsD1, E, and A, and E2F1 and separated by 10% SDS-PAGE. Left: Representative immunoprecipitation analysis, with control proteins (blockingpeptides) in the last lane. Right: Chemiluminescence analysis showing mean values � SD of 5-9 rats, normalized to control proteins (arbitrary units).TK test, F344: C vs. N and H, at least P � .05 for all genes. Wistar: C vs. N and H, and BN: C vs. H, at least P � .05 for c-Myc, Cyclin D1, andE2F1, not significant for Cyclins E and A. F344 vs. Wistar and BN, N and H, P � .001 and C, not significant for all genes.

Fig. 4. Representative reproduction of Cyclin D1-CDK4, Cyclin E-CDK2, and E2F1-DP1 complexes in normal liver (C), neoplastic nodules(N), and HCCs (H) of F344 and Wistar rats. N and H were induced by ISprotocol in F344 rats, and by ISS protocol in Wistar rats, as described inthe Materials and Methods. Proteins were immunoprecipitated by anti-bodies against Cyclin D1, Cyclin E, and E2F1, separated by SDS-PAGE,and then probed, together with blocking peptides, with antibodiesagainst CDK4, CDK2, and DP1. Enhanced chemiluminescence was usedto visualize the immune complexes. Upper panel: Representative immu-noprecipitation analysis, with control proteins (blocking peptides) in thelast lane. Lower panel: Chemiluminescence analysis showing meanvalues � SD of 3 rats, normalized to control proteins (arbitrary units). TKtest, F344: C vs. N and H, at least P � .05 for all complexes. Wistar: Cvs. N and H, not significant. F344 vs. Wistar and BN, N and H, at leastP � .05 and C, not significant for all genes.


3-fold increases in p16INK4A expression occurred in neo-plastic lesions of both Wistar and BN rats. In contrast,p27KIP1 gene expression did not undergo any change inliver lesions of all rat strains.

DiscussionOur results show that genetically determined resistance

to rat hepatocarcinogenesis is associated with the inabilityof neoplastic nodules to progress to full malignancy. Nod-ules with atypical patterns and moderately/poorly differ-entiated HCCs were relatively few or absent in theresistant rats. This was associated with a decrease in DNAsynthesis and an increase in nodule remodeling. This sit-uation apparently is not influenced by interstrain differ-ences in the initiation of carcinogenesis. Carcinogenmetabolism was not evaluated in this study, but low pro-gression capacity of nodules occurred in rat strains exhib-iting both decreases (Wistar) and increases (BN) in lesionnumber. Treatment of Wistar rats with 2 AAF cycles (ISSprotocol) resulted in significant increases in nodule num-ber and growth rate, but not in HCC incidence and mul-tiplicity above the values found in BN rats subjected to IS.

These observations suggest that genetic factors respon-sible for resistance to hepatocarcinogenesis influence themolecular mechanisms responsible for regulation of

growth and differentiation in initiated liver cells. CyclinsD1, E, and A overexpression suggests the existence ofenhanced G1 and S phase progression in nodules andHCCs of susceptible rats. These lesions exhibit c-myc am-plification and overexpression.6,27,28 c-Myc is a transcrip-tional regulator that controls cell cycle progressionthrough complex and not yet completely clear mecha-

Fig. 6. (A) RT-PCR and (B) immunoprecipitation analyses of p16INK4A

and p27KIP1 from normal liver (C), neoplastic nodules (N), and HCCs (H)of F344, Wistar, and BN rat strains. N and H were induced by IS protocolin F344 and BN rats, and by ISS protocol in Wistar rats, as described inthe Materials and Methods. RT-PCR products were separated by electro-phoresis into 6% polyacrylamide gel/10% glycerol. Size of PCR productswas evaluated by comparing ethidium bromide–stained RT-PCR productswith the fragment size marker, after electrophoresis in 2% agarose gel.Sample proteins and blocking peptides were coimmunoprecipitated byantibodies against p16INK4A and p27KIP1 and separated by 10% SDS-PAGE. Upper panels: Representative experiments. Lower panels: (A)Quantitative Instant Imager analysis showing mean radioactive counts �SD of 5-7 rats, normalized to Gapdh values (relative amounts). (B)Chemiluminescence analysis showing mean values � SD of 5-9 rats,normalized to control proteins (arbitrary units). TK test, RT-PCR, and IP:F344 rats, C vs. N and H, not significant for both genes. Wistar, C vs. Nand H, and BN, C vs. H, P � .001 for p16INK4A, not significant for p27KIP1.F344 vs. Wistar and BN, p16INK4A: at least P � .05 for N and H, notsignificant for C. p27KIP1: not significant for C, N, and H.

Fig. 5. CDK4 activity in whole homogenates of normal liver (C),neoplastic nodules (N), and HCCs (H) of F344, Wistar, and BN ratstrains. N and H were induced by IS protocol in F344 and BN rats, andby ISS protocol in Wistar rats, as described in the Materials and Methods.CDK4 activity was measured as incorporation of 32P from [�32P]ATPinto GST-pRb fusion protein, by using the protein immunoprecipitated byantibodies against CDK4 as a source of enzyme. Controls withoutantibody, sample, or substrate gave negative results and were notincluded in the Figure. Upper panel: Representative Instant Imageranalysis; Lower panel: Quantitative analysis of 3 rats. TK test, F344: Cvs. N and H, P � .001. F344 vs. Wistar and BN, N and H, P � .05, Cnot significant.


nisms. It may induce or repress Cyclin D1 gene29,30 or mayregulate Cyclin D1 posttranslationally.31 However, thislatter mechanism is not of primary importance in ourexperimental system, in which Cyclin D1 is up-regulatedat both mRNA and protein levels. Cyclin D1 is inducedby growth factors, such as transforming growth factor �(TGF-�),32 and overexpressed in chemically induced pre-neoplastic and neoplastic liver lesions.9 Cyclin D1 up-regulation also occurs in neoplastic liver of c-myc/tgf-�double transgenic mice and in c-myc transgenes.10 Theselatter mice also overexpress tgf-�, although at a lower levelthan double transgenes. Thus, combined overexpressionof c-myc and tgf-� in liver lesions of susceptible rats andmice could contribute to Cyclin D1 up-regulation.32

E2F1 may regulate c-Myc levels, and vice versa,10,33,34 anda positive feedback loop between E2F and Cyclins D1 andE also has been postulated.35 c-Myc may participate in theactivation of Cyclin E-CDK2 complexes by sequesteringp27KIP1, thus blocking the p27KIP1 binding to these com-plexes.36,37 In apparent contrast with the behavior of G1cyclins, no variation in CDK4 and CDK2 expression oc-curs in nodules and HCCs. Increases in Cyclin D1-CDK4 and Cyclin E-CDK2 complexes are consistentwith the existence of a CDK excess in liver cells38 andsuggest that cyclins are limiting components of com-plexes. Based on these observations, it may be concludedthat c-myc amplification and overexpression and up-reg-ulation of TGF-�, in chemically induced neoplastic liverlesions of susceptible rats, are implicated in deregulationof the pRb-E2F pathway, resulting in fast G1 progressionand G1-S transition. The mechanisms leading to CyclinA up-regulation in nodules and HCCs of susceptible ratsare unknown. Regulation of Cyclin A at the transcrip-tional level by E2F1 has been reported.35 However, thismechanism cannot explain Cyclin A mRNA overexpres-sion in these lesions. Further studies are necessary to clar-ify this point. Overexpression of Cyclins D1, E, and Aand of CDK4 have been observed recently in preneoplas-tic and neoplastic liver of Long Evans Cinnamon (LEC)rats.39 These rats exhibit oxidative damage, consequent toabnormal hepatic copper accumulation caused by muta-tion of the Atp7b gene that regulates copper transport.40

This indicates that predisposition to hepatocarcinogen-esis may result in the disruption of the pRb-E2F pathwayindependently of the genetic predisposing mechanism.

In liver lesions of genetically resistant rat strains, low orno increase in c-myc, Cyclins, and E2F1 expression occurs.This does not reflect interstrain differences in gene expres-sion unrelated to the susceptibility to hepatocarcinogen-esis because of the absence of differences at the level ofnormal liver. Therefore, it seems that c-myc and, eventu-ally, cell cycle key genes represent downstream genes, the

expression of which is regulated by Hcr loci. These locialso may influence p16INK4A gene expression, although themechanism (either genetic or epigenetic) involved is notclear. Up-regulation of p16INK4A, without changes inp27KIP1 expression, occurs in neoplastic liver nodules ofresistant rats. p16INK4A binds only the CDK4/6 proteins,thus inhibiting Cyclin D1-CDK4/6 complex assembly.41

Because p27KIP1 only binds Cyclin-CDK complexes, de-creases in cyclin D1-CDK4 complexes in nodules shouldincrease the pool of free p27KIP1, thus allowing more p27to bind and inhibit Cyclin E-CDK2 complexes.

Detailed genetic analysis of BN, F344, and BFF1 ratsled to the identification of 3 Hcs loci controlling nodulevolume1 (De Miglio et al., unpublished results). c-myc islocated at the Hcs1 locus, on chromosome 7 that oftenundergoes allelic imbalance in HCCs induced in the sus-ceptible LFF1 progeny4 and is syntenic to a human chro-mosome 8 segment in which frequent duplications occurin HCCs.9 c-myc is a candidate susceptibility gene, or mayrepresent a target of susceptibility genes located at Hcsloci. These genes should not be active in BN rats or theiractivity is modified by resistance genes. c-myc amplifica-tion is absent or infrequent in these rats,5 and in a ge-nome-wide study, allelic imbalance could not be found inHCCs of BFF1 rats.8 So far, 7 Hcr loci, inhibiting nodulegrowth and HCC incidence, were identified in BN rats1

(De Miglio et al., unpublished observations). Because nointerstrain differences in growth of neonatal liver, regen-erating adult liver, and liver weight of adult rats may beseen (data not presented), it may be inferred that resis-tance genes are activated as a consequence of carcinogentreatment. Our results indicate that active Hcr loci restrictthe expression of G1 and S phases regulatory genes andup-regulate the p16INK4A oncosuppressor gene in initiatedcells. However, we cannot exclude the possibility thatp16INK4A is directly involved as a resistance gene.

Up-regulation of c-MYC, associated with overexpres-sion of CYCLIN D1, CYCLIN E, and CYCLIN A genes,occur in human HCCs.16-18,42 Cyclin E and E2F1 up-regulation, associated with pRb under-regulation, occursin HCV core stable transfectant Rat-1 cell lines.43,44 De-regulated Cyclin E may induce chromosome instability,45

a common feature of human HCCs, that can be observedin HCCs of susceptible mice and rats,4,46 but not in resis-tant rats.8 Moreover, down-regulation of p16INK4A andp27KIP1 occurs in human HCCs19,20 as a consequence, atleast for p16INK4A, of promoter methylation.20,47 Thismakes a difference from the rat and mouse hepatocarci-nogenesis models, in which p16INK4A overexpression inc-myc/tgf-� transgenic mice,10 and no change in suscepti-ble rats were found. Genetic susceptibility to hepatocar-cinogenesis has not been studied yet in humans, because


of the rarity of familial clusters of HCC.48 However, hu-man HCC progression and prognosis are positively cor-related with Cyclins D1, E, and A expression andnegatively correlated with p16INK4A expression.17,18,49

This report provides a molecular mechanism of geneticpredisposition to HCC and suggests that genetic suscep-tibility to liver cancer may also control the prognosisthrough its influence on the progression capacity of liverlesions.

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Cell cycle deregulation in liver lesions of rats with and without genetic predisposition to hepatocarcinogenesis