Journal of Gastroenterology and Hepatology

(2003)

18,

80–91

Blackwell Science, LtdOxford, UKJGHJournal of Gastroenterology and Hepatology0815-93192003 Blackwell Publishing Asia Pty Ltd

182902

Liver pathology in HBx transgenic miceR Lakhtakia et al.

10.1046/j.0815-9319.2002.02902.xOriginal Article8091BEES SGML

Correspondence: Professor SK Panda, Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar,New Delhi 110029, India. Email: pandask@hotmail.com

Accepted for publication 25 July 2002.

HEPATITIS AND HEPATOCELLULAR CARCINOMA

Hepatocellular carcinoma in a hepatitis B ‘x’ transgenic mouse model: A sequential pathological evaluation

RITU LAKHTAKIA,* VIJAY KUMAR,

†

HONEY REDDI,

†

MEERA MATHUR,* SIDDHARTHA DATTAGUPTA* AND SUBRAT KUMAR PANDA*

*

Department of Pathology, All India Institute of Medical Sciences and

†

Virology Group, International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India

Abstract

Background

: The introduction of transgenic technology has made it possible to study the steps ofcarcinogenesis and directly establish the link between viral subgenomic fragments and specific types ofcancer. Research directed at hepatitis B virus (HBV)-related carcinogenesis has benefited from thistechnology. We present a detailed pathological evaluation of the sequential steps of hepatocarcinogenesisin a hepatitis B ‘x’ (HBx) transgenic mouse model. In this model, the transgene incorporates the regionencoding amino acids 58–154 of the HBV X protein and the murine c-myc gene. This model demon-strated changes in the liver from birth with foci of multicentric dysplasia evolving into nodules and overthepatocellular carcinoma between 20 and 28 weeks.

Methods and Results

: The hepatocytes were mitotically active and showed increased proliferativecapacity soon after birth, with exponential increase thereafter. This was accompanied by a high rate ofapoptosis, which later declined as the tumors developed. Other functional and immunophenotypic char-acteristics included a high c-myc expression in the neoplastic lesions, no alteration in p53 expression,and no alteration in the expression of hepatic enzymes except for diffuse expression of succinic dehy-drogenase.

Conclusion

: The entire process illustrates the disturbances of cell growth and death because of the col-laborative influence of HBx and c-myc genes that result in the development of hepatocellular carcinomaafter a prolonged latent period.© 2003 Blackwell Publishing Asia Pty Ltd

Key words

: experimental hepatocarcinogenesis, hepatitis B virus, transgenesis.

INTRODUCTION

Hepatocellular carcinoma (HCC) is the fourth com-mon cancer affecting mankind.

1

There is sufficient epi-demiological,

2–6

clinical,

7,8

and experimental evidence

9

linking hepatitis B virus (HBV) infection to liver cellcancer. Approximately 400 million people worldwidehave HBV infection and are at a heightened risk ofdeveloping liver cancer.

10

Although the association ofHBV with HCC is known, the exact pathogenic mech-anisms and sequential alterations in hepatocytes leadingto malignant transformation remain under investiga-tion. Both insertional mutagenesis and transactivationhave been implicated in carcinogenesis.

11

Several sub-

genomic fragments of HBV, namely X, S (surface), andpre-S, have been found to have transactivator capabil-ity.

12

Of these, the most extensively investigated candi-date has been the X protein that has demonstrablypotent transactivating properties.

13

With the intro-duction of transgenic technology in the research arma-mentarium,

14

several models of ‘X’ transgenic micedeveloping HCC at a higher frequency have beendescribed.

3,15–18

We have developed a HBV X-transgenic mousemodel (containing a bi-cistronic truncated X-c-mycfragment), which develops liver cell cancer between 20and 28 weeks of life. A preliminary pathological evalu-ation of a transgenic model heterozygous for this tran-

Liver pathology in HBx transgenic mice

81

script had revealed development of liver tumors within12–24 weeks.

19

This study investigates the sequentialhistological and immunohistochemical alterations thatlead up to the development of liver cancer in a trans-genic model homozygous for the same transcript. Thismay help in predicting histological and immunohis-tochemical markers in the precancerous stages of car-cinogenesis in the liver of high-risk HBV-infectedindividuals leading to proactive monitoring and therapy.

METHODS

Transgenic animals

The experimental animals used in the present studyconsisted of a colony of transgenic mice created by theembryo microinjection technique.

14

The genome incor-porated a bi-cistronic novel DNA construct consistingof a segment of the hepatitis B virus X gene expressinga truncated X protein (X15, amino acids 58–154) alongwith the murine c-myc gene (US patent no. 6274788approved 14 August 2001). It has been demonstratedearlier

in vitro

that this X gene segment was sufficient toprovide the critical transactivating function.

13

The colony created initially expressed this constructin a heterozygous fashion. A preliminary pathologicalevaluation of the organs of these mice, as they aged,revealed development of progressive multifocal lesionsin the liver ranging from dysplastic foci to hepatocellularcarcinomas between 12 and 20 weeks of age.

19

Thisprompted us to undertake a detailed pathological studyof a homozygous model of the same construct to ana-lyze this unique form of HBV-X gene-induced trans-genic hepatocarcinogenesis. Polymerase chain reactionevaluation of the tails of new-born pups confirmedhomozygous expression of the genetic transcript intro-duced in these animals (data not shown).

Collection of tissues

The animals were killed at intervals as depicted inFig. 1. The intervals for sacrifice were planned based onour initial observations of the temporal evolution ofmacroscopic and microscopic lesions in transgenic ani-

mals heterozygous for the X15-myc transcript referredto above.

19

A minimum of three animals of either sexwere evaluated up to day 14 (D14), and a minimum oftwo animals of either sex from D28 onwards. A total of154 animals were studied. The entire liver was collectedfrom animals killed on days 1, 3, 7, 14, 21, 28, andthereafter at weekly intervals up to 12 weeks and fort-nightly up to 48 weeks. A complete autopsy wasperformed and all organs were collected for study inanimals aged day 1 and day 7, and weeks 4, 8, 12, 14,20, 28, 36, and 44. In addition, age- and sex-matchednon-transgenic control animals were also killed to serveas controls. Animals were cared for humanely in accor-dance with institutional guidelines.

Pathology of the tissues

A record of gross appearances of organs with specialattention to the liver and its weight was made. All tissuesfor routine histopathological evaluation were collectedin 10% buffered formalin. Part of the liver tissue was, inaddition, collected in 4% glutaraldehyde for electronmicroscopy and 5

¥

1 mm pieces were snap-frozen inliquid nitrogen and stored at

-

70

∞

C for study of liverenzymes, fat accumulation and glycogen storage. Allorgans were studied by using routine light microscopyfor morphological alterations and presence ofmetastases, if any. Microscopic alterations in the liverwere studied with the help of routine histological stains(as detailed below), and histochemical and enzyme his-tochemical stains. Immunohistochemical determinationof proliferative potential, oncogene expression and stro-mal changes was performed. Apoptosis detection wasconducted using the TdT-mediated dUTP Nick EndLabeling (TUNEL) technique. Electron microscopywas carried out to determine changes at the ultrastruc-tural level.

Light microscopy

The following staining procedures were used: (i) hema-toxylin and eosin stain for routine morphological eval-uation of architectural patterns and cytological details;(ii) special stains were used where indicated by spe-cific findings on HE sections. These included:

Reticulin

(Gordon and Sweet’s method):

20

Reticulin fibers werehighlighted by the reduced silver deposited on them.

Glycogen

(Periodic Acid Schiff technique, McManus):

20

Glycogen positivity was seen as magenta–pink granulesin the cytoplasm, which was diastase-sensitive.

Iron

(Perl’s):

20

Iron was seen as intracytoplasmic blue gran-ules.

Oil Red O

staining (Lillie 1954)

21

was performedon frozen sections to look for fatty change; (iii) enzymehistochemistry: Cryostat sections (5–6

m

m) of freshfrozen tissue and tissue fixed in 1% formal calciumfollowed by gum sucrose solution were stored at

-

20

∞

Cand later stained to detect alterations in expressionof liver enzymes.

Glucose-6-phosphatase

(Wachstein andMeisel (1956)),

22

Succinic dehydrogenase

(Nitrobluetetrazolium (NBT) method of Nachlas

et al

. (1957)

23

as modified by Horwitz

et al.

(1967)).

24

Adenosine tri-

Figure 1

Experimental design for the sequential collectionof liver and other tissues from transgenic mice (X-15/myc).Note: Liver was collected at all stages. All organs collected atthe stages are shown in shaded figures.

82

R Lakhtakia

et al.

phosphatase

(Wachstein and Meisel (1951)),

25

Alkalinephosphatase

(Calcium cobalt method of Gomori(1951)).

26

Acid phosphatase

(Lead nitrate method ofGomori (1941)

27

as modified by Desmet (1962),

28

Gamma-glutamyl transpeptidase

;

29

(iv) immunohis-tochemical staining was performed for the detection ofproliferating cell nuclear antigen (PCNA), p53 protein,c-myc and fibronectin.

The antigens were detected by using the Avidin–Biotin Complex (ABC) technique (Vectastain ABC kit,Peroxidase Mouse PK 4002, Goat PK 4005; VectorLaboratories, Burlingame, CA, USA) with 3,3

¢

diami-nobenzidine tetrahydrochloride (DAB; Sigma D-0426,St Louis, MO, USA) as chromogen, which yielded abrown product at the appropriate site.

Briefly, after deparaffinizing the sections, endogenousperoxidase was blocked with 3% H

2

O

2

-Methanol.Sections were then treated with normal serum fromthe same species in which the secondary antibodywas raised (to prevent non-specific binding), and thenincubated at room temperature (RT) overnight withprimary antibody in the dilutions noted below. Afterwashing in Tris buffer, the sections were incubatedsequentially first with secondary antibody (1 h) andthen with Avidin–biotin complex for 45 min. Finally,sections were stained with a chromogen (DAB 1 mg/mLTris buffer) to localize the brown product at the antigensite in the tissues.

The antigens studied were as follows:

Proliferatingcell nuclear antigen

(PCNA; Clone PC 10, Cat # SC-56,monoclonal mouse IgG; Santa Cruz Biotechnology,Santa Cruz, CA, USA). The antibody used was diluted1:50, with microwave antigen retrieval in citrate bufferpH 6. Nuclei expressing PCNA stained brown. ThePCNA Labelling Index (LI) was determined byexpressing the percentage of positive hepatocyte nucleiafter counting at least 1000 nuclei;

p53

(Clone FL 393G, Cat # SC-6243-G, polyclonal goat IgG; Santa CruzBiotechnology). Antibody dilution 1:100, with micro-wave antigen retrieval in citrate buffer pH 6. Theantibody detects the product of mutated p53 as abrown staining of the nuclei;

c-myc

protein (Clone C-8, Cat # SC-41, IgG monoclonal mouse IgG; SantaCruz Biotechnology). Antibody dilution 1:50, withmicrowave antigen retrieval in citrate buffer pH 6. Thec-myc expression can be detected as staining of thenucleus or cytoplasm;

Fibronectin

(Clone N-20, Cat #SC-6953, polyclonal goat IgG; Santa Cruz Biotechnol-ogy). Antibody dilution 1:50, with microwave antigenretrieval in citrate buffer pH 6. Fibronectin staining canbe seen in the extracellular stroma in the portal tractsand in the hepatocytes bordering the portal tracts.

Control non-transgenic mice livers and relevantpositive controls were used alongwith.

(v) Assessment of cell death (apoptosis) by TUNELassay. The TdT-mediated dUTP Nick-End Labeling(TUNEL) assay was used to study apoptosis by using afluorescein based kit, and apoptotic cells were detectedby using the fluorescent microscope (Promega Corpo-ration Apoptosis Detection System, Madison, WI,USA; Fluorescein Cat. No.G3250). Sections weredeparaffinized with xylene and rehydrated throughdescending grades of alcohol. Then after, fixing in 4%

methanol free formaldehyde in PBS, they were perme-abilized by proteinase K (20

m

g/mL) for 8–10 min andfixed again in formaldehyde. After equilibrating the tis-sue in equilibration buffer, sections were incubated in amixture of TdT enzyme (1

m

L), Nucleotide mix (5

m

L),and equilibration buffer (45

m

L) under plastic coverslipsat 37

∞

C for 60 min in a humidified chamber coveredwith aluminum foil. The reaction was terminated with0.3 mol/L sodium chloride and 0.03 mol/L sodium cit-rate, pH 7.0 (2

¥

SSC). Sections were then counter-stained with propidium iodide (1

m

g/mL) for 15 min.After washing the sections, they were mounted in gly-cerol gel and examined under a fluorescence micro-scope at 520 nm using a standard fluorescein filter.Fluorescein-12-dUTP is incorporated at the 3

¢

-OHends of fragmented DNA, resulting in localized greenfluorescence within the nucleus of apoptotic cells. Pro-pidium iodide stains both apoptotic and non-apoptoticcells red throughout the cytoplasm.

Electron microscopy

Liver tissue fixed in 4% glutaraldehyde and embeddedin EPON resin (Cat No. 18010; Pelco International,Redding, CA, USA) was stained with uranyl acetate-lead citrate, and ultrathin sections (60 nm) were exam-ined by using a Philips CM 10 Electron Microscope PW6020 (Philips, Eindovan, The Netherlands) for ultra-structural changes in the transgenic and control livers.

RESULTS

Gross examination

The only noteworthy changes were in the liver. All otherorgans studied over the observation period did not showany significant change compared with the controls.

The liver of the transgenic mice showed an increase insize and weight disproportionate to the age-matchedcontrols (Fig. 2). This was most noticeable after20 weeks of life. At 28 weeks, the liver started appearingnodular, congested and firm in consistency; the changes

Figure 2

A comparative graph showing the difference ofliver weights between transgenic mice (X-15/myc) and age-and sex-matched controls. (

▲

) Control, (

�

) test.

Liver pathology in HBx transgenic mice

83

assuming a florid form between 28 and 32 weeks whenmultiple tumor nodules were evident on the surface.On a cut section, the tumor nodules showed centralnecrosis (Fig. 3).

Histopathological changes in the liver

Light microscopic and ultrastructural features

On hematoxylin and eosin stained sections, the trans-genic mice livers showed an early disappearance of fociof extramedullary hematopoiesis (EMH) in the portaltracts by day 14, whereas the same was seen to persist inthe controls by the fourth week. The cytological changesin the hepatocytes started as early as day 3, when mito-sis could easily be recognized in the hepatocytes aroundthe central veins (while they were absent in the con-trols). From day 7, these hepatocytes showed pro-gressive nuclear enlargement, vesicular chromatin,prominent, single or multiple nucleoli, nuclear pleo-morphism, and typical and atypical mitosis. Virtuallyevery centrizonal area demonstrated these abnormali-

ties. The cytoplasm of these hepatocytes was clear up to4 weeks and later became more basophilic. Apoptoticbodies were seen scattered in the sinusoids. Approxi-mately 8–12 weeks of focal necrosis was seen as well.Between 4 and 12 weeks, Kupffer cells appeared pro-minent (Fig. 4a,b).

Architectural alterations were seen from week 4onwards. At first, there was an expansion of the hepaticlobules with a clustering of large numbers of centralveins in a given field with spaced out portal tracts at theperiphery. By 16–20 weeks, multiple dysplastic fociwere incorporated into distinct neoplastic nodules thatcompressed the surrounding liver parenchyma. Thenodules showed compressed reticulin at the periphery(Fig. 4c,d).

Between 24 and 32 weeks, there was an emergence ofa multifocal, well-differentiated, hepatocellular carci-noma with a trabecular pattern. The malignant hepato-cyte cords showed large pleomorphic nuclei withmultinucleation and macronucleoli. Atypical mitosesand necrosis were present. The adjoining compressedliver continued to display the cytological features of dys-plasia noted in the early stages, providing the unstablebackground against which the carcinoma had evolved(Figs 5a; 6a,b).

At the ultrastructural level, the appearances paral-leled those seen on light microscopy. At day 3, the hepa-tocytes appeared swollen with abundant glycogen andendoplasmic reticulum-mitochondrial complexes. Byday 14, nuclear enlargement was evident, some withabnormal contours, while the cytoplasm containednumerous mitochondria including abnormal forms.At week 4, the glycogen accumulation peaked withenlarged nucleii showing prominent nucleoli. Occa-sional apoptotic bodies were also visualized. Fromweeks 5–12, initial mitochondrial-ER complexes werevisualized along with mitochondria, but later onlyabnormal mitochondria remained. This was accom-panied by a gross reduction in glycogen. The nucleuswas the dominating feature of the cell with abundantheterochromatin and large nucleoli (Fig. 7a,b).

Histochemical changes

Periodic Acid Schiff (PAS) stain (with and without dia-stase) revealed abundant glycogen in the hepatocytesup to week 4, which then gradually diminished andremained periportal (zone 1) in location (where theleast alterations were seen morphologically on HE).Reticulin fibers were compressed at the periphery of theemerging neoplastic nodules from 20 weeks of age.There was no development of cirrhosis. There was mildmicrovesicular fatty change in the hepatocytes aroundthe central veins at weeks 4–5. Perl’s stain showed ironin the dysplastic hepatocytes around the central veins,but the tumor cells were negative.

In enzyme histochemical studies, the liver showed adiffuse staining pattern with no zonal distribution ofsuccinic dehydrogenase in contrast to controls, whichshowed a periportal distribution throughout all thestages (Fig. 5d). This was a reflection of the abundance

Figure 3

Gross appearance of the transgenic mice at38 weeks: An enlarged, congested, nodular liver is seen fillingup the abdominal cavity. A non-transgenic age-matched con-trol is seen on the left for comparison. Date of birth,20.09.1999; Date of sacrifice, 5.5.2000.

84

R Lakhtakia

et al.

of mitochondria seen on electron microscopy. Therewas no alteration in the expression of glucose-6-phos-phatase, acid and alkaline phosphatase and adenosinetriphosphatase as compared with controls during theevolution of the lesions. Re-emergence of

g

-glutamyltransferase was not seen in the lesions.

Expression of p53 and c-myc protein

The p53 expression (mutation) was not detected in anystage of the neoplastic transformation. C-myc expres-sion was detected predominantly in a cytoplasmic loca-tion from 4 to 8 weeks of life and it increased thereafter.It was more prominent in the neoplastic nodules com-pared with the surrounding liver (Fig. 5b).

Extracellular matrix proteins

Fibronectin staining in the extracellular stroma was notdifferent from the control liver being localized to theportal regions. However, focal intracytoplasmic posi-tivity was seen in the tumor cells.

Proliferation and cell death

Expression of proliferating cell nuclear antigen (PCNA)increased with increasing age, reaching a plateau at 14–20 weeks with PCNA LI ranging from 5.4% in the earlystages to 45.8% at 20 weeks (Controls

<

0.5%; Fig. 8).The pattern of positivity was diffuse up to day 21.Thereafter, the nuclear staining became darker, coarserand more focalized to the nuclei of hepatocytes aroundthe central veins (Fig. 9a,b).

Apoptosis, detected as green fluorescein-stained cellswas scattered among the hepatocytes in the livers ofmice aged D3 and this increased up to 20 weeks of age(Fig. 5c). Thereafter, it declined sharply and was absentin the neoplastic nodules. The control livers showedoccasional randomly distributed apoptotic cells.

Control animals

The non-transgenic control animals did not show anysignificant increase in weight and size (Figs 2,3) com-pared with the transgenic animals. There were no cyto-

Figure 4

Microscopic appearance of early hepatic lesions seen up to 20 weeks (a–d). (a) Centrizonal hepatocytes at day 14showing mild pleomorphism and occasional mitosis (HE

¥

10). (b) At 4 weeks, there was moderate variation in nuclear size andprominent nucleoli in the centrizonal hepatocytes. Mitosis were easily seen. Kupffer cells appear prominent (HE

¥

20). (c) Earlynodule formation at 16 weeks exhibiting cellular dysplasia and increased mitosis (HE

¥

10). (d) Reticulin condensation aroundthe nodules at 20 weeks (Reticulin

¥

10).

a b

c d

Liver pathology in HBx transgenic mice

85

architectural abnormalities on microscopic examinationwith no consequent tumor development. Animals incor-porating c-myc alone (without X) also did not developtumors. No significant enzymatic aberrations werenoted at the tissue level. Proliferation remained at aconstant low level (Fig. 8), and there was no alterationin the expression of p53 or c-myc (Table 1).

DISCUSSION

Transgenesis provides a unique opportunity to observegene regulation, mutagenesis and creation of animalmodels of human disease.

30

The advances made in thisfield over the last decade have yielded tremendousinsights into genomics and enabled accurate localizationand manipulation of genes in these models, with an aimto direct therapy.

31

For infections like hepatitis B virus(HBV) with limited host range, this has permitted thestudy of the natural course of infection and the induc-tion of neoplastic change.

32

It was the seminal report of Chisari

et al

. in 1989 thatinitiated the process of understanding how preneoplas-tic lesions in HBV-transgenic mice evolved, leading upto hepatocellular carcinoma.

33

However, over the nexthalf-decade, some workers failed to reproduce theseevents.

34,35

In recent times, investigators have reaf-firmed the veracity of observations by Chisari

et al.

andKoike

et al.

,3,15,33 explaining that the inability to pro-duce tumors in some models may be because of abelow-threshold level of X gene expression and absenceof liver specific promoters. In studies on HBV-inducedHCC in transgenic mouse models, the X gene hasemerged as the ‘culprit’ behind the genotypic and phe-notypic changes that culminate in the development ofHCC, with a smaller contribution by HBs.16,36 In mod-els incorporating the X gene, unlike those with envelopeproteins, there is a prominent absence of inflammationin the preneoplastic stages: signifying a direct contribu-tion of HBx to the carcinogenic process.37

Some of the leading studies on HBV-X transgenichepatocarcinogenesis have dealt with the timing ofappearance of tumors, morphological features of the

Figure 5 Microscopic appearance of hepatocellular carcinoma (HCC) (a–d). (a) An unencapsulated nodule of a well-differentiated HCC showing a trabecular pattern and compressing the surrounding dysplastic hepatocytes. Inflammatory cells,mostly lymphocytes, are seen at the interface (HE ¥ 10). (b) Neoplastic hepatocytes show strong c-myc positivity (immunohis-tochemistry (IHC) (DAB) ¥ 10). (c) Only occasional apoptosis is seen in the sinusoids between the neoplastic cell cords (TdT-mediated dUTP Nick-End Labeling ¥ 40). (d) Diffuse succinic dehydrogenase (SDH) positivity in the neoplastic liver cells withloss of zonal distribution. (SDH ¥ 4).

86 R Lakhtakia et al.

HCC that developed, X expression in the emerginglesions, proliferative activity, and DNA ploidy.3,16,17 Inthe model by Koike,3 80–90% mice developed tumorsat approximately 13 months of age. Hepatitis B x wasoverexpressed in the clear cell foci noted at 8 weeks; itsexpression directly correlating with tumor develop-ment.15 The 5-Bromo2-deoxy Uridine (BrdU) uptakewas increased. Ultimately, a multifocal well-differenti-ated HCC developed. The model by Kim et al. devel-oped lesions later (4 months), which were clear cells,glycogen-rich and strongly expressed HBx.16 However,a significant feature was the lack of proliferative activity.Adenomatous nodules developed at 8–10 months, andwas followed by a clear cell HCC and then death within11–15 months.16 The model by Terradillos et al.17 incor-porated X and c-myc, and this combination developedtumors 8–14 weeks earlier than if c-myc alone waspresent. Mitotic figures and apoptosis were noted at30 days when the architecture was still normal. The pro-liferation rate was sixfold higher, but the apoptotic rateremained the same throughout. Hyperplastic nodulesdeveloped at 80 days, and the HCC was similar to themodel by Koike. The workers support a two-hit theory

for the evolution of these lesions with the X gene pro-viding the initiating stimulus and other factors like alter-ations in c-myc protoncogene17 or disturbed signalingpathways38 completing the process of transformationto a malignant phenotype. The current study hasattempted to make a detailed morpho-immunopheno-typic study of the changes seen in the liver from birth todeath in a similar model incorporating multiple param-eters to understand the biofunctional changes in theliver before malignancy supervenes.

C-myc is a cellular proto-oncogene that can triggereither proliferative or apoptotic responses dependingupon the cellular context.17 While on the one hand HBV‘X’-induced carcinogenesis is linked to transactivationof c-myc and c-jun genes, X-gene is also known to

Figure 6 Microscopic appearance of hepatocellular carci-noma. (a) Hepatocellular carcinoma demonstrating nuclearpleomorphism, nucleomegaly with macronucleoli and pseu-donuclear inclusions (HE ¥ 40). (b) Reticulin enclosing cordsof neoplastic hepatocytes (Reticulin ¥ 10).

a

b

Figure 7 Electron microscopic appearances of liver. (a)Numerous mitochondria with megamitochondria at 16 weeks(Uranyl acetate–lead citrate ¥ 3400). (b) Large nucleus withmultiple nucleoli in a hepatocellular carcinoma at 32 weeks(Uranyl acetate–lead citrate ¥ 6300).

a

b

Liver pathology in HBx transgenic mice 87

potentiate the effect of c-myc-induced oncogenesis.Independent workers amply demonstrated this inbitransgenic models by combining c-myc with X trans-gene, tumor growth factor (TGF)-a or hepatocytegrowth factor (HGF) genes.17,39,40 We observeddevelopment of HCC between 12 and 20 weeks in atransgenic mouse model with heterozygous expressionof a truncated X/myc construct.19

The current model was therefore devised with ahomozygous expression of the same transgene for adetailed pathological evaluation of the hepatocarcino-genesis process. This had a potent effect: firstly, thetranscript being liver-specific, the changes seen at allages were diffuse and multifocal (comparable withthe successful transgenic models of Koike et al.3,15).Secondly, the proliferative stimulus was so strong thatmitotic activity was noticeable in our animals from day3 and increased exponentially with advancing age longbefore morphological, functional or immunophenotypicchanges were identifiable.

Despite the evidence of increased proliferative activ-ity and dysplastic changes in the hepatocytes surround-ing the central veins, the dysplasia-carcinoma sequencedeveloped only after 20 weeks (in contrast to theheterozygous model where tumors appeared after12 weeks). In this context, a noteworthy finding was thepattern of apoptosis (seen on HE and by the TUNELtechnique). While apoptosis was present in the earlystages, when the tumors developed, it rapidly disap-peared, whereas in the heterozygous model, it was loweven in the early stages and thus allowed tumors todevelop at 12–24 weeks.19 The peculiar control exer-cised by c-myc in balancing proliferation and apoptosisis possibly responsible for this phenomenon. AlthoughKoike et al. observed a latent period of 10 monthsbefore the adenoma–carcinoma sequence developed, inthe intermediate period, many of the ‘altered foci’showed evidence of multiple aneuploid populations andincreased bromodeoxyuridine (BrdU) uptake, indicat-ing an overall capacity of these early lesions to prolifer-ate in an unregulated fashion. However, they did not seeany apoptotic activity.3

The tumors in our model were well-differentiatedhepatocellular carcinomas, and did not show prominentlocal invasion or distant metastasis. There was no cir-rhotic phase, and alteration of framework (reticulin)became apparent only at 20 weeks with no significantchanges in the extracellular matrix (fibronectin).

A high-HCC yielding model of HBx transgenic mice(86%) has been recently devised by Yu et al., which pro-vides ample material for study.18 In HBs transgenicmice, interestingly, high tumor producer lineages showsustained labeling of S-phase hepatocytes from the stageof injury to the development of HCC, while low pro-ducer lineages do not show this after the initial stage ofinjury.41 As multiple risk factors, including activation ofproto-oncogenes like c-myc or growth factors like TGF-a, insulin-like growth factor-II (IGF-II)42 or hepatocytegrowth factor (HGF)39 are incorporated in to trans-genic models; the rates of proliferation are seen toincrease exponentially. Notably, in all these models,a concomitant increase in apoptosis is not seen—theimbalance probably providing the milieu for neoplastictransformation.

Figure 8 Proliferating cell nuclear antigen labeling index(PCNA LI) of hepatocytes of transgenic mice from birth todevelopment of hepatocellular carcinoma. (�) Control, �test.

Figure 9 Immunohistochemical detection of proliferatingcell nuclear antigen (PCNA) expression in transgenic micehepatocytes. (a) Coarse and focal nuclear staining of the cen-trizonal hepatocytes at 8 weeks for PCNA (PCNA (diami-nobenzidine tetrahydrochloride (DAB) ¥ 10). (b) Multiplehepatocyte nuclei showing PCNA positivity at the hepatocel-lular carcinoma stage at 32 weeks (PCNA (DAB) ¥ 10).

a

b

88 R Lakhtakia et al.

A significant presence of apoptosis has been reportedrecently in the transition between normal liver and thefinal development of HCC, at which point proliferationseems to overtake apoptosis, and neoplastic nodulesemerge.43 In this context, it is significant that, HBxincorporation along with c-myc is associated with atwofold increase in apoptosis. This not only con-tributes to HBV pathogenesis in the chronically infectedliver but also possibly favors the regeneration andaccumulation of genetic alterations, finally leading toliver cell transformation.40,44 In a growth hormonetransgenic mouse model of hepatocarcinogenesis, thenon-neoplastic liver and the dysplastic nodules

showed high levels of apoptosis keeping pace with pro-liferation, whereas in the tumor areas, this declinedabruptly.45

Other morphophenotypic changes on the road toneoplasia included a search into enzyme alterations. Incontrast to experimental models of chemical carcino-genesis, we could find no re-emergence of fetal enzymeslike GGT in the preneoplastic nodules. The only con-stant change in enzyme expression was a diffuse stainingfor SDH (a mitochondrial enzyme), which wasendorsed by the diffuse occupation of the hepatocytecytoplasm on electron microscopy (EM) by enlargedand abnormal mitochondria.

Table 1 Comparative pathological features of liver of X15/myc transgenic mice and non-transgenic controls

Features Transgenic mice Controls

Gross Progressive liver enlargement with sharpincrease in weight from 20 weeks. Nodularityat 28 weeks. Development of tumors grosslybetween 28 and 32 weeks

Normal liver size commensuratewith age, no nodularity or tumordevelopment

MicroscopicCyto-architectural changes Mitosis seen from day 3 and increasing dysplasia

of the centrizonal hepatocytes from day 7.Scattered apoptotic bodies in the sinusoids.Expansion of lobules from week 4 with microscopicnodules between 16 and 20 weeks. Hepatocellularcarcinoma (well-differentiated, multifocal) between24 and 32 weeks. No vascular invasion or invasionof adjoining organs seen. Adjoining liver showspersistent dysplastic foci

Normal lobular architecturemaintained No evidence ofhepatocyte dysplasia. Portal tracts normal

Ultrastructural changes Glycogen abundance until week 4. Thereafter, enlarged abnormal megamitochondria and large nucleus withprominent nucleolus

Glycogen and mitochondria(normal) in all stages. Nonucleomegaly or prominence ofnucleoli.

Histochemical alterations Strong PAS positive (diastase resistant) hepatocytes upto week 4 and decline thereafter with restriction toZone 1. Iron seen in dysplastic hepatocytes.Centrizonal microvesicular fatty change at weeks4 and 5. Diffuse SDH positivity, no alterations inacid and alkaline phosphatase, glucose-6-phosphatase, ATPase. No re-emergence of GGT.Reticulin compressed around emerging nodules at20 weeks. No frank cirrhosis. Fibronectin positivityin portal regions as in controls, focal positivity intumor cells

Hepatocytes glycogen rich with nodepletion except in old age inZones 2 and 3. No ironaccumulation or fatty change.Normal patterns of enzymelocalization and expression. Nore-emergence of GGT. Noincrease in reticulin. Normalfibronectin deposition inextracellular location in portaltracts

p53 and c-myc expression No p53 expression (mutation) detected by IHC. No p53 or c-myc expressionC-myc expressed in dysplastic hepatocytes and

stronger in tumor cells compared to adjoining liverProliferation (Proliferating

cell nuclear antigenLabeling Index;(PCNA LI) and celldeath (Apoptosis)

Increasing PCNA LI plateauing at 20–24 weeks (range5.4–45.8%). Pattern of staining diffuse up to week 3and then coarse and focalized to centrizonaldysplastic hepatocytes. Apoptosis prominent byTUNEL technique, peaking at 20 weeks anddeclining thereafter

PCNA LI < 0.5% Only randomoccasional apoptosis at all stages

ATPase, adenosine triphosphate; GGT, gamma glutamyl transpeptidase; IHC, immunohistochemistry; PAS, Periodic AcidSchiff stain; PCNA, proliferating cell nuclear antigen; SDH, succinic dehydrogenase; TUNEL, TdT-mediated dUTP Nick EndLabeling.

Liver pathology in HBx transgenic mice 89

There is a paucity of literature on ultrastructuralchanges in HCC-producing models of transgenic mice.Oval cell proliferation has been noted by using immu-noelectron microscopy showing transitional featuresbetween oval cells and hepatocytes.46 Singh et al. noteda progressive mitochondrial degeneration in their HBxtransgenic mice.19 In a rat transgenic model, thenodules showed less cytoplasmic differentiation thanmature hepatocytes with fewer mitochondria and lesserendoplasmic reticulum (ER). Focally, cells were stuffedwith mitochondria and the nuclei showed abundantheterochromatin. The tumors showed smaller cells, thecytoplasms of which were stuffed with mitochondria.47

We observed cytoplasmic glycogen abundance in theearly stages and numerous abnormal mitochondrialater. The nucleus with a prominent nucleolus domi-nated the cell as a whole.

Alterations in the p53 gene, that regulates the activityof the cell cycle machinery and acts as a transcriptionfactor, are perhaps the most common type of geneticlesions in human cancer. In human HCC, p53 expres-sion (mutated p53) is increased in the tumor and itssurrounding tissues and in regenerative nodules of cir-rhotic livers that have not yet developed HCC.48

Genetic analysis reveals an increasing loss of heterozy-gosity (LOH) for chromosome 17p (on which p53 geneis located), along with the abnormality of the Rb geneand other chromosomal mutations, with decreasing dif-ferentiation of the tumor substantiating the belief thattumor progression is accompanied by accumulatedgenetic changes.49 Transgenic mouse models of p53provide a unique opportunity to understand the conse-quences of its mutation. One such model containing thep53ser246 mutation (corresponding to p53ser249mutation in human HCC associated with HBV infec-tion), showed a large number of cells in the G1 phase,but did not show a difference in apoptosis rates.50 Whencombined with other predisposing factors like liverinjury, male gender and aflatoxin exposure, the tumor-igenic effect of p53 is greatly enhanced.51

Hepatitis B x has been reported to bind p53 proteinand inactivate it.35 Ueda et al. suggested that this bind-ing is more functional in the early stages, and p53 muta-tion appears much later in advanced stages of cancer.52

Although normal wild type p53 is related to the cells’apoptotic function, in the HBx transgenic model, adirect dose-dependent p53-independent method ofapoptosis has been seen.40 There was no p53 mutationin our model even in the late stages of carcinogenesis.This further endorses the view that it may be the resultrather than the cause of the neoplastic process, and thatp53 alterations, per se, had little role to play in govern-ing the cell cycle disturbances in this model.

With the availability of the transgenic mouse model,elucidation of pathways of carcinomatous changethrough preneoplastic stages will open newer vistas fortherapeutic manipulations. This has been exploredthrough experiments studying an antiviral drugbis(POM)PMEA against HBV infection,53 interleukin-12 in inhibiting HBV replication at hepatic and extra-hepatic sites54 and reduction in the expression of viralsequences, and c-myc overexpression by a-interferon,which prevented the development of hepatic dysplasia55

or intraperitoneal administration of phosphorothioateoligodeoxynucleotides, which decreased HBx expres-sion and development of preneoplastic lesions.56

This model has demonstrated the vagaries of the bio-logical evolution of neoplastic lesions, but also brings uscloser to understanding how therapeutic interventionsto control cell growth and death have the potential to tiltthe balance towards regression of early lesions defyingthe driving neoplastic stimuli. One of the major thrustareas in current transgene and liver cancer research willbe the development and identification of markers thatcan predict future cancer development in patients withchronic HBV and HCV infection. This would narrowdown a subgroup of patients who need a more intensivefollow up to detect and pre-empt malignant transfor-mation. Future transgenic models will be aimed atalteration of the genome directing subtle changes inmolecular pathways that will halt or alter preneoplasticprogression.

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