neu Transformed Rat Cholangiocytes Recapitulate Key Cellular and Molecular Features of Human Bile Duct Cancer

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erbB-2/neu Transformed Rat Cholangiocytes Recapitulate Key Cellular and Molecular Features of Human Bile Duct Cancer GUAN–HUA LAI,* ZICHEN ZHANG,* XUE–NING SHEN,* DEANNA J. WARD,* JENNIFER L. DEWITT,* SHAWN E. HOLT,*,‡ REBECCA A. ROZICH,§ DOUGLAS C. HIXSON,§ and ALPHONSE E. SIRICA* *Division of Cellular and Molecular Pathogenesis, Department of Pathology, ‡Departments of Genetics and of Pharmacology and Toxicology, Virginia Commonwealth University School of Medicine, Medical College of Virginia Campus, Richmond, Virginia; and the §Department of Medical Oncology, Rhode Island Hospital, Providence, Rhode Island

Background & Aims: Cholangiocarcinomas appear to arise from the malignant transformation of cholangiocytes lining the biliary tract. Because the development of an in vitro model of malignant transformation can provide a powerful new tool for establishing critical events governing the molecular pathogenesis of cholangiocarcinoma, we investigated the potential of achieving malignant transformation of cultured rat cholangiocytes in relation to aberrant overexpression of mutationally activated erbB-2/neu. Methods: Malignant neoplastic transformation was achieved after infection of the rat cholangiocyte cell line, designated BDE1, with the retrovirus Glu664-neu, containing the transforming rat erbB-2/neu oncogene. Results: Compared with untransformed control cells, malignant transformants carrying the activating erbB-2/neu mutation prominently overexpressed p185neu receptor protein, which was phosphorylated strongly at its major autophosphorylation site at tyrosine 1248. Moreover, erbB-2/neu transformation of BDE1 cells resulted in increased telomerase activity, upregulation of cyclooxygenase-2 with overproduction of prostaglandin E2, enhanced phosphorylation of mitogenactivated protein kinase and of serine/threonine kinase Akt/PKB, overexpression of vascular endothelial growth factor, and increased mucin 1 messenger RNA expression. Only erbB-2/neu transformants were tumorigenic when transplanted into isogeneic rats, yielding a 100% incidence of tumors closely resembling human desmoplastic ductal cholangiocarcinomas in their morphology. Malignant cholangiocytes in the tumors were strongly immunoreactive for biliary cytokeratin 19, p185neu, and cyclooxygenase-2. Conclusions: This unique malignant transformation model recapitulates key molecular features of the human disease and appears to be well suited for testing novel molecular therapeutic strategies against cholangiocarcinoma.

holangiocarcinoma is an insidious cancer with a high rate of mortality and only limited treatment options, which in the majority of cases have proven to be ineffective in eliciting long-term survival responses. The classic diagnosis is well-differentiated to moderately dif-

C

ferentiated desmoplastic ductal adenocarcinoma, although less common histologic variants also are recognized including papillary adenocarcinoma, intestinal-type adenocarcinoma, mucinous adenocarcinoma, and biliary cystadenocarcinoma.1–3 Approximately 40%–70% of cholangiocarcinomas occur at the liver hilum at or near the bifurcation of the right and left hepatic ducts, and 5%–15% of these tumors form within liver.2 The establishment of novel experimental models of cholangiocarcinoma that recapitulate cellular and molecular properties of the human disease not only can facilitate studies directed toward elucidating the cellular and molecular pathogenesis of cholangiocarcinoma, but, just as compelling from the standpoint of clinical relevance, have the potential to serve as preclinical platforms for testing new therapeutic strategies aimed at exploiting select molecular targets. Such potential targets observed to be overexpressed in cases of human cholangiocarcinoma include telomerase, mucin 1 (MUC1), cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), and ErbB-2/Neu.2 In this context, a strong positive correlation between quantitative immunostaining intensities for plasma membrane ErbB-2/Neu and cytoplasmic COX-2 in human cholangiocarcinomas and related risk conditions has been reported.4 ErbB-2/Neu has been shown further to be overexpressed in the neoplastic glandular epithelium of furan- and thioacetamide-induced intestinal-type cholangiocarcinomas in rat liver,5,6 with COX-2 also having been found in the furan model to be up-regulated strongly in malignant cholangiocytes overexpressing constitutively tyrosineAbbreviations used in this paper: Akt, serine/threonine kinase Akt/ PKB; CK19, cytokeratin 19; COX-2, cyclooxygenase-2; Ct, threshold cycle value; MAP, mitogen-activated protein; MUC1, mucin 1; PCR, polymerase chain reaction; RT, reverse-transcription; VEGF, vascular endothelial growth factor. © 2005 by the American Gastroenterological Association 0016-5085/05/$30.00 doi:10.1053/j.gastro.2005.10.010

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phosphorylated ErbB-2/Neu.7,8 The relationship between ErbB-2/Neu overexpression and COX-2 up-regulation in biliary tract cancers is supported further by the finding that BK5.ErbB-2A transgenic mice overexpressing wild-type rat c-erbB-2/neu transgene preferentially developed papillary adenocarcinomas of the gallbladder, showing increased levels of COX-2 messenger RNA (mRNA) and protein.9 Although these in vivo rodent models clearly reflect select features of certain subtypes of human biliary tract cancers (ie, intestinal type and papillary adenocarcinomas), they do not recapitulate the histopathologic features of typical desmoplastic ductal cholangiocarcinomas. Ductal cholangiocarcinomas develop in the Syrian hamster model, but, unfortunately, this cancer model has not been well characterized in terms of its molecular pathogenesis. There have been a limited number of immortalized but nontumorigenic rodent and human cholangiocyte cell lines published.10 –13 BDE1 is a novel cell line that was derived as a clonogenic population from an epithelial cell outgrowth of bile duct fragments isolated from normal Fischer 344 rat liver and subsequently precultured in a rat-tail collagen gel.14 In contrast to rat liver stem-like cell lines, BDE1 cells were shown to express morphologic and phenotypic markers consistent with those of liver cholangiocytes.14,15 We now report on the malignant transformation of cultured BDE1 cells as a result of constitutive overexpression of tyrosine-phosphorylated ErbB-2/Neu after retroviral infection of these bile duct– derived epithelial cells with mutationally activated neu oncogene, the rat homologue of human erbB-2. Our analysis of the transformants strongly supports the establishment of a novel model of cholangiocyte malignant transformation that recapitulates key cellular and molecular characteristics of the human disease, and that appears to be well suited to test relevant molecular targeting strategies.

Materials and Methods Materials Dulbecco’s modified Eagle medium, Geneticin (G-418 sulfate), KaryoMAX Giemsa stain stock solution, Gurr buffer tablets, and TRIZOL reagent were purchased from Invitrogen Corp (Carlsbad, CA). Colchicine, fetal bovine serum, insulin, penicillin-streptomycin, transferrin, trypsin-ethylenediaminetetraacetic acid solution, and Hanks’ balanced salt solution were obtained from Sigma-Aldrich Co. (St. Louis, MO). Bacto agar was purchased from Fisher Scientific (Pittsburgh, PA). DakoCytomation target retrieval solution (10⫻ concentrate) and DAKO DAB (diaminobenzidine tetrahydrochloride) chromogen tablets were purchased from DAKO Corp. (Carpinteria, CA). Zymed Trypsin Histo-Kit for immunohistostaining was

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obtained from Zymed Laboratories, Inc (South San Francisco, CA), and appropriate Vectastain Elite avidin-biotin-peroxidase immunostaining kits were purchased from Vector Laboratories (Burlingame, CA).

Methods Culture conditions, retroviral infection, and selection. Under standard conditions, untransformed BDE1 rat cholangiocytes were cultured at 37°C in 95% air/5% CO2 on plastic substratum in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, .1 ␮mol/L insulin, 5 ␮g/mL transferrin, 100 U/mL penicillin, and 100 ␮g/mL streptomycin. At in vitro passage 98, when at ⬃70% confluence, these cells were infected by a retroviral vector carrying a complete complementary DNA copy of the transforming rat erbB-2/neu oncogene (Glu664neu) together with the neomycin (G418) resistance gene, as previously described for cultured WB-F344 rat liver stem-like cells.7,8,16 Glu664neu contains a point mutation in the transmembrane domain substituting a glutamine for the normal valine at residue 664, leading to full oncogenic activation of the rat erbB-2/neu gene.17 After selection in standard medium containing 500 ␮g/mL G418, surviving cell outgrowths were harvested by trypsinization and determined to be strongly positive for the expression of p185neu. The harvested cells were pooled and the resulting cell line was designated BDEneu. A control cell line, designated BDEneo, also was established in a comparable manner by infecting BDE1 cells at passage 98 with the retroviral vector expressing only the neomycin resistance gene. Both the BDEneu and BDEneo cell lines tested negative for mycoplasm as determined by enzyme immunoassay using the Mycoplasm Detection Kit purchased from Roche Applied Science (Penzberg, Germany). DNA amplification and sequencing of the rat erbB-2/neu transmembrane domain. Polymerase chain reaction (PCR) was used to amplify the transmembrane domain spanning nucleotides 1931–2105 of the rat erbB-2/neu gene expressed by BDEneu cells using the following PCR primers: upstream primer sequence: 5=-ACTTCCTGTGTGGATCTGGAT-3=; downstream primer sequence: 5=-AGCCTACGCATCGTATACTT-3=. Genomic DNA (1.0 ␮g) was subjected to 40 cycles of amplification (denaturing: 94°C, 1 min; annealing: 56°C, 1 min; extension: 72°C, 2 min per cycle) using the Perkin Elmer Gene Amp PCR System 9600 (Boston, MA), as previously described.5 Amplified PCR product, separated by electrophoresis on 2.0% agarose gel, was purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) and then sequenced in an ABI 3700 DNA Analyzer (Applied Biosystems, Foster City, CA). Plasmid pJRneu carrying the rat erbB-2/neu oncogene mutationally activated at nucleotide 2012 of the transmembrane domain served as the positive control, as previously described,5 whereas pSVneu, carrying wild-type rat erbB-2/neu (kindly provided by Dr. Mien-Chie Hung of the University of Texas M.D. Anderson Cancer Center, Houston, TX), served as the nonmutated control. Each sample was analyzed in duplicate.

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Chromosome analysis and telomeric repeat amplification protocol. Untransformed BDE1 cells at in vitro passage 98 and BDEneu transformed cells at an early passage after retroviral infection were subjected to karyotype analysis as previously described.18 Briefly, 1-day-old cultures at approximately 80% confluence were exposed to .1 ␮g/mL colchicine for 4 to 6 hours at 37°C. They then were harvested, pelleted by centrifugation, and placed in a hypotonic solution of KCl (.068 mol/L) for 15 minutes, followed by a 10-minute fixation at room temperature in a freshly prepared solution of methanol/glacial acetic acid (3:1 vol/vol). Fixed cells were pelleted, washed 3⫻ in fixative (final wash at 4°C), and then resuspended gently in 1.0 mL ice-cold fixative. Three or 4 drops of the fixed cells then were pipetted onto ice-cold microscope slides, dried, and stained with a Giemsa staining solution composed of 3.0 mL KaryoMax Giemsa stain stock solution in 48.5 mL Gurr buffer, pH 6.8 (Invitrogen Corp). For each cell line, a total of 50 metaphase chromosome spreads were counted. Telomerase activity was determined by the telomeric repeat amplification protocol, as previously described,19 with slight modification. Telomeric repeat amplification protocol was performed on cell extracts corresponding to 1000 cells prepared from cultured BDE1, BDEneo, and BDEneu cells, respectively. The C611B ChC tubular adenocarcinoma cell line derived from a furan-induced rat cholangiocarcinoma18 was used as a positive control. Telomeric repeat amplification protocol reactions were extended for 30 minutes at room temperature. The extension products were PCR amplified for 27 cycles, and resolved on a 10% polyacrylamide gel. Separated gel products then were exposed to a phosphoimaging cassette (Molecular Dynamics, Sunnyvale, CA) and directly scanned and analyzed using ImageQuant Software (Molecular Dynamics). A 36-bp internal standard was used to verify successful amplification and to serve as a standard for relative quantitation. Relative (semiquantitative) telomerase activity, termed Q, was calculated using the ratio of the intensity of the 6-bp telomerase-specific ladder to the intensity of the 36-bp internal standard. Prostaglandin E2 assay. Prostaglandin E2 (PGE2) production from arachidonic acid (30 ␮mol/L), added in serum-free Dulbecco’s modified Eagle medium, was assayed as previously described20 in medium collected from cultures of 2 ⫻ 106 BDE1, BDEneo, and BDEneu cells, respectively, by using the Prostaglandin E2 EIA Kit-Monoclonal (catalog no. 514010) purchased from Cayman Chemical (Ann Arbor, MI). Total cell protein per culture dish was measured using DC protein assay reagents (catalog no. 500-0116) purchased from Bio-Rad Laboratories (Hercules, CA). Each assay was performed in triplicate, with final PGE2 concentrations elaborated into medium calculated as pg/␮g total cell protein. Western blotting and primary antibodies. Western blot analysis of total protein in cell lysates comparably prepared from cultured BDE1, BDEneo, and BDEneu cells was performed as previously described5,8,20 using each of the following primary antibodies: (1) Neu (C18), sc-284, an affinity-

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purified rabbit polyclonal antibody raised against an epitope mapping at the carboxy terminus of human p185neu, and previously determined to be cross-reactive with rat cholangiocarcinoma p185neu5,8; (2) p-Neu (Tyr 1248)-R, sc-12352-R, an affinity-purified rabbit polyclonal antibody raised against an epitope corresponding to a short amino acid sequence containing phosphorylated tyrosine 1248 of human p185neu; (3) COX-2 (M19), sc-1747, an affinity-purified goat polyclonal antibody raised against a peptide mapping to the carboxy terminus of rat COX-2, and previously shown to be reactive with rat cholangiocarcinoma COX-28,20; (4) VEGF (A20), sc-152, an affinity-purified rabbit polyclonal antibody raised against a peptide mapping at the amino terminus of human VEGF; (5) actin (C-11), sc-1615, an affinity-purified goat polyclonal antibody raised against an epitope mapping at the carboxy terminus, identical to human, rat, and mouse peptide sequences; (6) affinity-purified rabbit polyclonal phosphop44/42 mitogen-activated protein (MAP) kinase (Thr202/ Tyr204) antibody (#9101S); (7) affinity-purified rabbit polyclonal p44/42 MAP kinase antibody (#9102); (8) affinitypurified rabbit polyclonal phospho-Akt (Ser 473) antibody (#9271); and (9) affinity-purified rabbit polyclonal Akt antibody (#9272). Antibodies 1–5 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), whereas antibodies 6 –9 were from Cell Signaling Technology (Beverly, MA). Densitometry measurements of individual protein bands normalized to ␤-actin were performed on triplicate Western blot samples and analyzed using Quantity One 1-D analysis software (Bio-Rad). Real-time reverse-transcription PCR analysis of ErbB-2/Neu, COX2, and MUC1. Total RNA was extracted using the SV Total RNA Isolation System purchased from Promega Corp (Madison, WI) according to the manufacturer’s instructions. Oligonucleotide primers and probes were designed using the Primer Express 1.5 software program (Applied Biosystems) and synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). The specific primer and probe sequences used in our real-time PCR analyses were as follows: glyceraldehyde-3-phosphate dehydrogenase (Gene bank accession #BC059110), forward primer: 5=- AAC CTG CCA AGT ATG ATG ACA TCA -3=, reverse primer: 5=- TGT TGA AGT CAC AGG AGA CAA CCT -3=, and probe sequence: 5=TGG TGA AGC AGG CGG CCG A -3=; ErbB2/Neu (Gene bank accession #X03362), forward primer: 5=- GAG ACT GAT GGC TAT GTT GCT C -3=, reverse primer: 5=- ATT CTT CCC AGG AGA GAG AGT C -3=, and probe sequence: 5=-AGA GGT TCA GCC TCA GCC TCC TTT AA -3=; COX-2 (Gene bank accession #AF233596), forward primer: 5=- GAC TGT ACC CGG ACT GGA TTC T -3=, reverse primer: 5=- TTC AGC GGT AAT TTG ATT CTT GTC-3=, and probe sequence: 5=- CGG TGA AAA CTG TAC TAC GCC GAG ATT CC -3=; and MUC1 (Gene bank accession # XM_342281), forward primer: 5=- CGG AAC CAC CCA TTT AAT TCT TC -3=, reverse primer: 5=- CCG TTA AAA ACC TGC AGA AAC AA -3=, and probe sequence: 5=- TGG AAG ACC CCA GCT CCC GCT -3=. All 4 probes described earlier

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were labeled at the 5= end with the reporter dye 6-Fam, whereas the quencher dye BHQ-1 was linked to the 3= end. One-step reverse transcription (RT)-PCR reactions were performed in separate tubes using the TaqMan One-Step RTPCR Master Mix Reagents Kit purchased from Applied Biosystems. Total RNA (2.5 ng) was subjected to real-time RTPCR using the Mx3000 P real-time PCR System from Stratagene (La Jolla, CA). The cycling parameters were as follows: reverse transcription at 48°C for 30 minutes; AmpliTaq activation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds; and annealing/extension at 60°C for 1 minute. Relative quantification of gene expression was determined using the 2⫺⌬⌬Ct method,21,22 where Ct is the threshold cycle value. All samples were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase, calculated by the formula ⌬Ct ⫽ Cttarget gene ⫺ Ctglyceraldehyde-3-phosphate dehydrogenase, with the target genes being ErbB2/Neu, COX-2, and MUC1, respectively. The N-fold increase in ErbB2/Neu, COX-2, and MUC1 mRNA expressed in BDEneu cells compared with that of BDEneo control cells was calculated using the following formula: 2⫺(⌬Ct BDEneu ⫺ ⌬Ct BDEneo). Each real-time RT-PCR experiment was performed in triplicate. Anchorage-dependent growth curves, anchorageindependent growth, and tumorigenicity assays. Anchorage-dependent growth curves were determined from hemocytometer counts of trypsinized BDEneu and parent BDE1 cells that had been plated initially at 6000 cells in 35-mm plastic wells coated with rat-tail type 1 collagen. In each case the cells were maintained under standard medium conditions and cell counts were made on triplicate cultures per time point. Cell population doubling times were determined from the linear phase of the growth curve. Anchorage-independent growth was assayed as previously described8 by seeding 1500 BDEneu (compared with BDE1) cells in culture medium containing .3% agar over a .6% soft agar layer in tissue culture wells. At the end of 2 weeks of culture, the total number of colonies formed per culture well were counted under phase contrast. Two separate animal studies were conducted in the Department of Pathology of Virginia Commonwealth Univer-

sity School of Medicine to evaluate tumorigenicity, using protocols approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University. In the first study, 2 individual tumorigenicity experiments were conducted in which young adult Fischer 344 male rats (Harlan, Indianapolis, IN) each were inoculated with 2 ⫻ 106 BDEneu or control BDEneo cells, subcutaneously in their right inguinal region, as previously described.20 In the second animal study, 4 ⫻ 106 BDEneu cells or control BDEneo cells suspended in .1 mL Hanks’ balanced salt solution were inoculated using a 26-gauge needle inserted into the proximal common bile duct near the liver hilus and directed toward the left hepatic duct into the liver of Fischer 344 male rats. At death, both tumor incidence and wet weights were determined. All tumors formed at either the subcutaneous or intrahepatic sites were subjected to routine histopathologic examination and evaluated in formalin-fixed, paraffin-embedded sections by using antigen retrieval combined with the avidin-biotin-peroxidase complex method for immunohistochemical staining of cytokeratin 19 (CK19), ErbB-2/Neu, ErbB-2/NeuPtyr 1248, and COX-2, and histochemically for mucin by the mucicarmine method, according to minor modifications of previously described methods.4,5,18 Primary antibodies, used at 1:100 and 1:200 dilutions, for immunohistochemical staining included HER2/ErbB-2 antibody (#2242) from Cell Signaling Technologies, p-Neu (Tyr 1248)-R (sc-12352-R) and COX-2 (M19) antibody from Santa Cruz Biotechnology, and CK19 antibody (NCL-CK19) from Novocastra Laboratories, Ltd (Newcastle upon Tyne, UK). No immunostaining was observed in negative control tissue sections in which either phosphate-buffered saline was substituted for the primary antibody (CK19, ErbB-2/Neu) or in those in which the primary antibody (ErbB-2/Neuptyr1248, COX-2) was neutralized by preabsorption with a specific blocking peptide. Additional experimental details are given in the footnotes to Table 1 and in the legend to Figure 7. Statistical analysis. Mean values ⫾ SD were calculated from pooled data from repeat experiments. The Student

Table 1. Tumorigenicity of BDEneu Transforments Compared With BDEneo Control Cells Experiment no

Number of rats per group

Cell line

Cell transplantation site

Mean animal weight (g) ⫾ SD at death

Tumor incidence, %

Mean tumor weight (g) ⫾ SD

Histologic type

1 1 2 2 1= 1=

5 6 5 6 6 6

BDEneu BDEneo BDEneu BDEneo BDEneu BDEneo

Subcutaneous Subcutaneous Subcutaneous Subcutaneous Intrahepaticc Intrahepaticc

249.38 ⫾ 15.77 329.58 ⫾ 12.09b 206.00 ⫾ 13.83 316.10 ⫾ 24.96b 160.75 ⫾ 22.73 213.77 ⫾ 12.37

100 0 100 0 100 0

1.58 ⫾ .23a 0 1.52 ⫾ .71a 0 7.46 ⫾ 2.13d 0

Ductal carcinoma — Ductal carcinoma — Ductal carcinoma —

bearing 1.0- to 1.5-cm diameter tumors formed at the subcutaneous inoculation site were killed 1 month after cell transplantation of 2 ⫻ 106 BDEneu cells per rat. bRats subcutaneously inoculated with 2 ⫻ 106 BDEneo cells killed 3 months after initial cell transplantation. cRats were killed 1 month after intrahepatic transplantation of 4 ⫻ 106 BDEneu or BDEneo cells. d Total combined mean net weight of livers plus BDEneu tumor was 15.52 ⫾ 3.34 g, whereas that of the BDEneo nontumor control livers was 9.16 ⫾ 1.24 g. aRats

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A

B

C

BDEneu

pJRneu

pSVneu

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Figure 1. (A) DNA sequence analysis of genomic DNA from rat BDE1 cells infected retrovirally with Glu664neu (BDEneu), showing a transversion point mutation at nucleotide 2012 (arrow) corresponding to that determined for (B) plasmid pJRneu carrying the erbB-2/neu gene showing the same mutation at the nucleotide 2012 hot spot (positive control). (C) Corresponding DNA sequence of wild-type rat erbB-2/neu gene carried in plasmid pSVneu (nonmutated control).

PCR followed by direct DNA sequencing was used to ascertain if the BDEneu cell line contained the activating point mutation at nucleotide 2012 hot spot of retrovirally introduced rat Glu664neu, altering the transmembrane domain of p185neu.23 Figure 1A shows the presence of the expected transversion activating mutation5,23 at nucleotide 2012 of erbB-2/neu in amplified genomic DNA from cultured BDEneu cells. Sequencing of the positive control (pJRneu) amplified from the 3= end using the downstream primer validated the presence of the A ¡ T transversion mutation (or corresponding T ¡ A mutation from 5=, using upstream primer) at the nucleotide 2012 hot spot corresponding to the transmembrane domain of mutationally activated rat erbB-2/neu (Figure 1B), compared with the wild-type sequence shown in Figure 1C. Karyotype analysis showed the untransformed parent BDE1 cell line and the derived BDEneu cell line have similar patterns of aneuploidy, with approximately 79% of the total metaphase spreads analyzed in each case having chromosome counts ranging from 61 to 64 (Figure 2). However, although retroviral infection of the BDE1 cells at in vitro passage 98 with mutationally activated rat erbB-2/neu did not alter modal chromosome number markedly, the resulting BDEneu transformants showed significant increases in both anchorage-dependent growth in collagen-coated wells and in their ability to proliferate and form colonies in soft agar compared with untransformed parent BDE1 cells (Figure 3). Moreover, as shown in Figure 4A, the relative telomerase activity of cultured BDEneu cells was determined after normalization to be enhanced markedly over that of both the parent BDE1 and BDEneo controls. Previously we reported that COX-2 is up-regulated strongly in both rat C611B ChC cholangiocarcinoma

A. 40 % of Total Metaphase Spreads Analyz ed

Results

cells constitutively overexpressing tyrosine-phosphorylated p185neu and in erbB-2/neu-transformed WB-F344 rat liver stem-like cells, with a concomitant overproduction of PGE2 from arachidonic acid being detected in the culture medium.7,8 Similarly, PGE2 production from arachidonic acid was found to be increased significantly (P ⱕ .001; n ⫽ 3) in the BDEneu cell cultures over basal

35

BDE1

30 25 20 15 10 5 0 52

54 58

59 61 62 63

64 65 66

Chromosome Number

B 35

% of Total Metaphase Spreads Analyzed

2-tailed t test was used to determine the P value, with a P value of .05 or less considered significant.

30

BDEneu

25 20 15 10 5 0

57

59

60 61

62

63

64 65 66

69

Chromosome Number Figure 2. (A) Karyotype analysis of cultured parent BDE1 cells compared with that of (B) the BDEneu cell line after retroviral infection with Glu664neu and G418 selection. For each cell line, chromosome counts were made on a total of 50 metaphase spreads.

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x33

A C

1.6

BDE1

*

BDEneu

1.4

Cell number ( 10 6)

x33

B

1.2 1 0.8 0.6 0.4 0.2 0 0

1

2

3

4

5

6

7

Number of Cell Colonies in Soft Agar

Days in Culture

140

D

*

120 100 80 60 40 20 0 BDE1

PGE2 levels produced by BDE1 and BDEneo control cell cultures, respectively (Figure 4B). Figure 5 is a representative composite of our Western blot analyses of cultured BDEneu cells compared with BDE1 and BDEneo control cells. As shown by our quantitative densitometry data, p185neu expression was increased significantly in BDEneu cells over that of the BDEneo and BDE1 cells, respectively. Perhaps even

BDEneu

8

9

Figure 3. (A) Representative phasecontrast photomicrograph showing the epithelial nature of BDEneu cells cultured to confluence on a plastic substratum. (B) Phase-contrast photomicrograph showing typical anchorage-independent colonies of BDEneu cells formed in soft agar at 2 weeks after initial cell seeding of 1500 cells. (C) Comparative anchorage-dependent growth curves for BDEneu cells vs parent BDE1 cells cultured on rat-tail type 1 collagencoated plastic wells. Each value represents the mean ⫾ SD from individual cell counts made on triplicate cultures per time point. Cell population doubling time: BDEneu ⫽ 18.9 hours; BDE1 ⫽ 21.3 hours. (D) Differences in the number of cell colonies formed after 2 weeks of anchorage-independent growth of BDEneu cells in soft agar compared with that of BDE1 cells. Each value represents the mean ⫾ SD from cell colony counts made on 3 separate cultures per cell line. *P ⱕ .001.

more significant is our finding that p185neu overexpressed in the BDEneu cells strongly is immunoreactive for autophosphorylation at its Tyr 1248 site (Figure 5), whereas p185neu-pTyr 1248 essentially was not detectable in BDEneo or in BDE1 cells, which do not harbor the Glu664neu gene. Of further significance is the fact that COX-2 protein was expressed strongly in the BDEneu cells only (Figure 5) when compared with the BDEneo

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36-bp Internal Standard Q: 1

1 11 5 -

B Pro stag lan d in E 2 in m ed iu m (p g / µ g )

BD BDE1 E BD ne E o C6 ne 11 u ly B si C s h bu C ffe r

A

2053

20

* 15 10 5 0 BDE1

BDEneo

BDEneu

Figure 4. (A) Representative telomeric repeat amplification protocol assay showing markedly increased telomerase activity expressed by cultured BDEneu cells compared with that of the BDEneo and BDE1 cell lines. Note that the baseline Q value (see Materials and Methods section for explanation of Q value) determined for BDEneo control cells was unchanged from that of parent BDE1 cells. The C611B ChC rat cholangiocarcinoma cell line served as a positive control. (B) PGE2 overproduction from arachidonic acid by cultured BDEneu cells compared with PGE2 elaborated into the medium by comparably maintained BDE1 and BDEneo control cells. Each value represents the mean ⫾ SD from individual measurements made on 3 separate cultures per cell line. P ⱕ .001 compared with BDEneo and BDE1.

and BDE1 controls, consistent with our findings for PGE2 production shown in Figure 4B. In addition, as also shown in Figure 5, a modest but significant increase in VEGF protein expression was observed in cultured BDEneu cells over that detected by Western blotting in both the BDEneo and parent BDE1 controls. The BDEneu, BDEneo, and BDE1 cell lines each showed comparable protein bands in Western blots for ␤-actin, p44/42 MAP kinase, and pAkt. In contrast, positive immunoreactivity for phosphorylation of p44/42 MAP kinase was barely detectable in the control BDEneo and BDE1 cells, but prominently shown by the BDEneu cells (Figure 5). The cultured BDEneu cells also showed a significantly increased level of Akt phosphorylation compared with that of the cultured BDEneo and BDE1 cells (Figure 5). Figure 6 shows representative real-time RT-PCR amplification plots for cultured BDEneu cells compared with BDEneo cells. Compatible with our Western blot data, BDEneu cells were found to express mean levels of ErbB-2/Neu, COX-2, and MUC1 apoprotein mRNA relative to glyceraldehyde-3-phosphate dehydrogenase that each were significantly greater (P ⱕ .001; n ⫽ 3), respectively, than those expressed by BDEneo control cells cultured under comparable conditions. Based on our calculations derived from Ct values, we determined the cultured BDEneu cells to express ErbB-2/Neu mRNA at a mean relative level that was 14.2-fold ⫾ 2.7-fold higher than that expressed by BDEneo control cells. The mean relative levels of COX-2 mRNA and of MUC1

mRNA apoprotein were calculated to be increased by 38.4-fold ⫾ 3.4-fold and 4.23-fold ⫾ .16-fold, respectively, over that expressed by the BDEneo cells. The ultimate indicator of malignant neoplastic cell transformation is the demonstration of the ability of the transformed cells to give rise to malignant tumors when transplanted into suitable host animals. As shown in Table 1, the BDEneu cells produced ductal carcinomas (Figure 7) at a 100% incidence when transplanted either subcutaneously or orthotopically into isogenic Fischer 344 young adult male rats. In sharp contrast, no tumors developed in any of the recipient animals comparably transplanted with BDEneo control cells, even after 3 months after initial cell transplantation into the subcutaneous site and at 1 month after orthotopic transplantation into liver, when the experiments were terminated. It also is particularly noteworthy that orthotopic transplantation of BDEneu cells by inoculation into the left hepatic bile duct resulted in rapidly growing invasive cancer, which in each case (n ⫽ 6) was localized preferentially to the left/median liver lobes (Figure 7A) and that also produced biliary obstruction at the hepatic hilus. Furthermore, all of the BDEneu liver tumors were observed to be associated with extrahepatic metastases to the abdominal mesentery and kidneys (data not shown). In contrast, tumors that formed at the subcutaneous site were seen to be more circumscribed in their growth than the hepatic tumors and did not show evidence of distant metastasis.

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Discussion In this study we used mutagenically activated erbB-2/neu to achieve in vitro malignant transformation of a cholangiocyte cell line initially established as a clonogenic outgrowth from a bile duct fragment isolated from normal adult rat liver. We report malignant transformation of cholangiocytes in vitro, and as such provide a unique model for investigating relevant pathways associated with the cellular and molecular pathogenesis of cholangiocarcinoma. More-

BDEneu-ErbB2/Neu BDEneo-ErbB2/Neu BDEneu-GAPDH BDEneo-GAPDH

Fluorescence (dRn)

A

Mean protein band density ratios relative to b –actin. 0.15

BDEneo 0.14

BDEneu 0.78* (P ⱕ 0.05)

p185neu-ptyr 1248

0.27

0.37

0.97* (P ⱕ 0.02)

p72/74 COX-2

0.0001

0.02

1.57* (P ⱕ 0.0001)

p44/42 MAP Kinase-P

0.06

0.001

1.17* (P ⱕ 0.005)

p42/44 MAP Kinase

2.27

2.50

2.20 *†

p60 phospho- Akt

0.18

0.17

1.29* (P ⱕ 0.02)

p60 Akt

2.78

2.57

2.24 *†

p21 VEGF

0.44

0.45

0.76* (P ⱕ 0.005)

Cycles

B Fluorescence (dRn)

BDE1 p185neu

* n=3 † NS = not significant

Figure 5. Representative Western blot analysis showing relevant altered protein expression and phosphorylation profiles of BDEneu cells compared with those of the BDE1 and BDEneo control cell lines. RIPA, no-cell buffer (negative) control. The mean protein-band density ratios reflect the mean band intensity for each protein of interest, as determined by densitometry, relative to that of corresponding ␤-actin, which served as the internal housekeeping standard.

Cycles

C Fluorescence (dRn)

Histopathologic evaluation of both the subcutaneous and intrahepatic tumors formed from the BDEneu cells showed them all to be desmoplastic ductal carcinomas, without evidence of hepatocyte differentiation. Phenotypically, the neoplastic ductal epithelial components of these tumors, whether subcutaneous or intrahepatic, strongly and uniformly were immunoreactive for biliary CK19, plasma membrane ErbB-2/Neu, and cytoplasmic COX-2, respectively, but only minimally positive for mucin histochemical staining (Figure 7). Moreover, the positive immunoreactivity shown by the malignant cholangiocytes for ErbB-2/Neuptyr1248 (Figure 7D) indicated that this oncoprotein is being expressed in its tyrosine-phosphorylated state, compatible with our Western blot data shown in Figure 5.

BDEneu-COX-2 BDEneo-COX-2 BDEneu-GAPDH BDEneo-GAPDH

BDEneu-MUC1 BDEneo-MUC1 BDEneu-GAPDH BDEneo-GAPDH

Cycles

Figure 6. Representative real-time RT-PCR amplification plots for (A) ErbB-2/Neu, (B) COX-2, and (C) MUC1 apoprotein mRNA levels compared with glyceraldehyde-3-phosphate dehydrogenase mRNA expressed in cultured BDEneu cells relative to corresponding mRNA levels for these gene products expressed in BDEneo control cells.

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CK19

RT RT

* a

b

A

ErbB-2/Neuptyr 1248

ErbB-2/Neu

X66

C

X132

D Mucicarmine

COX-2

E

X132

B

X66

F

X132

Figure 7. (A) Gross pathology of an invasive tumor formed in the liver of a Fischer 344 rat at 1 month after orthotopic cell transplantation of 4 ⫻ 106 BDEneu cells inoculated via the left hepatic bile duct compared with control liver transplanted with BDEneo cells. Arrows point to left liver lobe, which was bisected to expose the intrahepatic tumor. Note the irregular margins of the mass-forming tumor, which has replaced much of the normal liver tissue of the left hepatic and median lobes. The right liver lobe (RT) did not show evidence of tumor, but the hepatic hilus (*) at the level of the left hepatic bile duct was replaced by tumor. (B) No tumor was detected in liver comparably transplanted with BDEneo cells. (B–E) Representative photomicrographs showing strong uniform positive immunoreactivity of neoplastic ducts for biliary CK19, plasma membrane ErbB-2/Neu, ErbB-2/Neu immunoreactive for phosphotyrosine1248, and cytoplasmic COX-2, respectively, in desmoplastic ductal carcinoma developed in rats transplanted with BDEneu cells. (F) The low level of mucicarmine histochemical staining of mucin (red) present in neoplastic ducts of ductal carcinoma originated from transplanted BDEneu cells.

over, based on our presented findings, this model has the potential of serving as a powerful preclinical platform for testing selective molecular therapeutic targeting strategies against gene products aberrantly overexpressed in human cholangiocarcinomas, including activated p185neu, COX-2, and human telomerase reverse transcriptase (hTERT).2 Both the untransformed BDE1 and transformed BDEneu cells exhibited comparable degrees of aneuploidy, but only the BDEneu cells were observed to show significantly enhanced anchorage-independent growth in soft agar, which correlated with a 100% incidence of tumorigenicity. These data suggest that aneuploidy, which is a reliable indicator of

genetic instability, was in itself insufficient for tumorigenicity of BDE1 cells. Rather, it is evident from our results that introducing mutationally activated erbB-2/neu into aneuploid nontumorigenic BDE1 cells provided a critical molecular triggering event culminating in full-blown malignant neoplastic transformation. Notably we found that p185neu overexpressed in the BDEneu cells was strongly immunoreactive for constitutively phosphorylated Tyr 1248. Tyr 1248 is known to be a major autophosphorylation site at the carboxyl-terminal domain of ErbB-2/Neu protein that is correlated with the transforming potential of mutationally activated erbB-2/ neu.24 This, in turn, leads to enhanced downstream signal-

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ing through the RAS-Raf-p44/42 MAP kinase and Akt pathways, which is consistent with our Western blot data showing prominent phosphorylation of p44/42 MAP kinase and p60 Akt only in the BDEneu cells and not in either BDE1 or BDEneo controls in which phosphorylated Tyr 1248 essentially was not detected. The association between ErbB-2/Neu overexpression and COX-2 up-regulation previously suggested in rodent models of cholangiocarcinogenesis7–9 is reinforced strongly by our Western blot and real-time RT-PCR data showing dramatic increases in COX-2 protein and mRNA being expressed in only the BDEneu cell transformants overexpressing activated ErbB-2/Neu compared with the BDE1 and BDEneo controls. The significant overproduction of PGE2 by cultured BDEneu cells over that of BDE1 and BDEneo cells further emphasizes the functional relationship between erbB-2/neu transformation and COX-2 induction in the neoplastically transformed cholangiocytes, and likely plays an important role in the oncogenic process. That the BDEneu cells also show a prominent increase in their telomerase activity further suggests that ErbB-2/Neu overexpression and activation coupled to COX-2 up-regulation and increased PGE2 production also may be acting in a complementary manner to regulate telomerase expression in transformed cholangiocytes, thereby likely contributing to their tumorigenic phenotype. The desmoplastic ductal carcinomas formed from BDEneu cells after cell transplantation into isogenic Fischer 344 rats show morphologic features that are quite distinct from those of intestinal-type cholangiocarcinomas typically induced in the furan5,18 or thioacetamide6 rat models of cholangiocarcinogenesis, and those shown by human intestinal-type cholangiocarcinoma.1,3 In contrast to both rat and human intestinal-type cholangiocarcinomas, which show goblet cell metaplasia and abundant secretion of histochemically detected mucin,3,5,8 the BDEneu tumors did not contain metaplastic goblet cells nor did they show evidence of prominent mucin secretion by mucicarmine staining. Rather, the CK19-positive neoplastic ductal structures characteristic of the BDEneu tumors more closely resembled native biliary ducts than mucin-secreting metaplastic glands, recapitulating histopathologic features commonly observed in human ductal cholangiocarcinomas that more typically occur at the hepatic hilus of human liver. Here, it is also important to point out that unlike chemical or spontaneously transformed WB-F344 rat liver epithelial stem-like cells, which have been shown to give rise to a spectrum of diverse tumor types including hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, and sarcomas when transplanted subcutaneously into syngeneic rats,25,26 neo-

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plastically transformed BDEneu cells yielded only ductal carcinomas in our in vivo subcutaneous and hepatic tumorigenicity assays. Thus, it appears evident that rat BDEneu cells distinctly are different in their differentiation potential than are malignant transformed WBF344 rat liver stem-like cells being more compatible with that of a cell type that has committed to differentiate only along the cholangiocyte lineage. Because ErbB-2/Neu overexpression and COX-2 upregulation are characteristic features of furan-induced rat cholangiocarcinomas,5,7,18 and also were shown in a majority of well-differentiated human cholangiocarcinomas,4 our finding that the neoplastic ductal structures of the BDEneu tumors are strongly immunoreactive for both plasma membrane ErbB-2/Neu and for cytoplasmic COX-2 further support our view that overexpression of activated ErbB-2/Neu together with up-regulation of COX-2 reflect molecular alterations that appear to be common to both human and rat cholangiocarcinogenesis in general7 and not reflective of a specific cholangiocarcinoma subtype. Moreover, it is interesting that erbB-2/ neu transformation of BDE1 cells resulted in an increased expression of MUC1 mRNA over that expressed in nontumorigenic BDEneo cells. Human cholangiocarcinomas27,28 have been shown frequently to express membrane-bound MUC1, which has been correlated with both tumor progression and poor prognosis.28,29 On the other hand, MUC-2, an intestinal-type secreted mucin, has been associated with less aggressive cholangiocarcinoma and improved outcome.28,29 In this context, molecular analysis of specific mucin apoproteins expressed in furan-induced intestinal-type rat cholangiocarcinoma compared with more native ductal carcinoma derived from BDEneu cells now clearly seems to be warranted. The orthotopic tumorigenicity model described in this study has the real potential of being developed into a powerful preclinical platform for assessing new targeted therapies against intrahepatic cholangiocarcinoma recapitulating key cellular and molecular features of human bile ductal carcinoma. We are now focusing our efforts on the further development and characterization of this unique in vivo model with the anticipation that it will prove to be a powerful new instrument for the preclinical testing of novel adjuvant treatments against this insidious malignant disease.

References 1. Longnecker DS, Terhune PG. Carcinogenesis and pathology of carcinomas in the pancreas— comparison with the biliary tract. In: Sirica AE, Longnecker DS, eds. Biliary and pancreatic ductal epithelia-pathobiology and pathophysiology. New York: Marcel Dekker, Inc., 1997:527–562.

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2. Sirica AE. Cholangiocarcinoma: molecular targeting strategies for chemoprevention and therapy. Hepatology 2005;41:5–15. 3. Bae JY, Park YN, Nakanuma Y, Lee WJ, Kim JY, Park C. Intestinal type cholangiocarcinoma of intrahepatic large bile duct associated with hepatolithiasis—a new histologic subtype for further investigation. Hepatogastroenterology 2002;49:628 – 630. 4. Endo K, Yoon B, Pairojkul C, Demetris AJ, Sirica AE. ERBB-2 overexpression and cyclooxygenase-2 up-regulation in human cholangiocarcinoma and risk conditions. Hepatology 2002;36: 439 – 450. 5. Radaeva S, Ferreira-Gonzalez A, Sirica AE. Overexpression of C-NEU and C-MET during rat liver cholangiocarcinogenesis: a link between biliary intestinal metaplasia and mucin-producing cholangiocarcinoma. Hepatology 1999;29:1453–1462. 6. Yeh C-N, Maitra A, Lee K-F, Jan Y-Y, Chen M-F. Thioacetamideinduced intestinal-type cholangiocarcinoma in rat: an animal model recapitulating the multi-stage progression of human cholangiocarcinoma. Carcinogenesis 2004;25:631– 636. 7. Sirica AE, Lai G-H, Endo K, Zhang Z, Yoon B. Cyclooxygenase-2 and ERBB-2 in cholangiocarcinoma: potential therapeutic targets. Semin Liver Dis 2002;22:303–313. 8. Lai G-H, Zhang Z, Sirica AE. Celecoxib acts in a cyclooxygenase2-independent manner and in synergy with emodin to suppress rat cholangiocarcinoma growth in vitro through a mechanism involving enhanced Akt inactivation and increase activation of caspases-9 and -3. Mol Cancer Ther 2003;2:265–271. 9. Kiguchi K, Carbajal S, Chan K, Beltrán L, Ruffino L, Shen J, Matsumoto T, Yoshimi N, DiGiovanni J. Constitutive expression of ErbB-2 in gallbladder epithelium results in development of adenocarcinoma. Cancer Res 2001;61:6971– 6976. 10. Grubman SA, Perrone RD, Lee DW, Murray SL, Rogers LC, Wolkoff LI, Mulberg AE, Cherington V, Jefferson DM. Regulation of intracellular pH by immortalized human intrahepatic biliary epithelial cell lines. Am J Physiol 1994;266:G1060 –G1070. 11. Paradis K, Le ONL, Russo P, St-Cyr M, Fournier H, Bu D. Characterization and response to interleukin 1 and tumor necrosis factor of immortalized murine biliary epithelial cells. Gastroenterology 1995;109:1308 –1315. 12. Hreha G, Jefferson DM, Yu C-H, Grubman SA, Alsabeh R, Geller SA, Vierling JM. Immortalized intrahepatic mouse biliary epithelial cells: immunologic characterization and immunogenicity. Hepatology 1999;30:358 –371. 13. Maruyama M, Kobayashi N, Westerman KA, Sakaguchi M, Allain JE, Totsugawa T, Okitsu T, Fukazawa T, Weber A, Stolz DB, Leboulch P, Tanaka N. Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT. Transplantation 2004;77:446 – 451. 14. Yang L, Faris RA, Hixson DC. Long-term culture and characteristics of normal rat liver bile duct epithelial cells. Gastroenterology 1993;104:840 – 852. 15. Yang L, Faris RA, Hixson DC. Phenotypic heterogeneity within clonogenic ductal cell populations isolated from normal adult rat liver. Proc Soc Exp Biol Med 1993;204:280 –288. 16. Jou Y-S, Layhe B, Matesic DF, Chang C-C, deFeijter AW, Lockwood L, Welsch CW, Klaunig JE, Trosko JE. Inhibition of gap junctional intercellular communication and malignant transformation of rat liver epithelial cells by neu oncogene. Carcinogenesis 1995;16: 311–317.

erbB-2/neu TRANSFORMATION OF CHOLANGIOCYTES

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17. Bargmann CI, Weinberg RA. Oncogenic activation of the neuencoded receptor protein by point mutation and deletion. EMBO J 1988;7:2043–2052. 18. Lai G-H, Sirica AE. Establishment of a novel rat cholangiocarcinoma cell culture model. Carcinogenesis 1999;20:2335–2339. 19. Elmore LW, Turner KC, Gollahon LS, Landon MR, Jackson-Cook CK, Holt SE. Telomerase protects cancer-prone human cells from chromosomal instability and spontaneous immortalization. Cancer Biol Ther 2002;1:391–397. 20. Zhang Z, Lai G-H, Sirica AE. Celecoxib-induced apoptosis in rat cholangiocarcinoma cells mediated by Akt inactivation and Bax translocation. Hepatology 2004;39:1028 –1037. 21. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2⫺⌬⌬CT method. Methods 2001;25:402– 408. 22. Kinnman N, Francoz C, Barbu V, Wendum D, Rey C, Hutcrantz R, Poupon R, Housset C. The myofibroblastic conversion of peribiliary fibrogenic cells distinct from hepatic stellate cells is stimulated by platelet-derived growth factor during liver fibrogenesis. Lab Invest 2003;83:163–173. 23. Bargmann CI, Hung M-C, Weinberg RA. Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell 1986;45:649 – 657. 24. Akiyama T, Matsuda S, Namba Y, Saito T, Toyoshima K, Yamamoto T. The transforming potential of the c-erbB-2 protein is regulated by its autophosphorylation at the carboxyl-terminal domain. Mol Cell Biol 1991;11:833– 842. 25. Tsao M-S, Grisham JW. Hepatocarcinomas, cholangiocarcinomas, and hepatoblastomas produced by chemically transformed cultured rat liver epithelial cells: a light and electron-microscopic analysis. Am J Pathol 1987;127:168 –181. 26. Hooth MJ, Coleman WB, Presnell SC, Borchert KM, Grisham JW, Smith GJ. Spontaneous neoplastic transformation of WB-F344 rat liver epithelial cells. Am J Pathol 1998;153:1913–1921. 27. Jan Y-Y, Yeh T-S, Yeh J-N, Yang H-R, Chen M-F. Expression of epidermal growth factor receptor, apomucins, matrix metalloproteinases and p53 in rat and human cholangiocarcinoma—appraisal of an animal model of cholangiocarcinoma. Ann Surg 2004;240:89 –94. 28. Higashi M, Yonezawa S, Ho JJL, Tanaka S, Irimura T, Kim YS, Sato E. Expression of MUC1 and MUC2 mucin antigens in intrahepatic bile duct tumors: its relationship with a new morphological classification of cholangiocarcinoma. Hepatology 1999;30: 1347–1355. 29. Tamada S, Goto M, Nomoto M, Nagata K, Shimizo T, Tanaka S, Sakoda K, Imai K, Yonezawa S. Expression of MUC1 and MUC2 mucins in extrahepatic bile duct carcinomas: its relationship with tumor progression and prognosis. Pathol Int 2002;52:713–723.

Received May 9, 2005. Accepted September 7, 2005. Address requests for reprints to: Alphonse E. Sirica, MS, PhD, Division of Cellular and Molecular Pathogenesis, Department of Pathology, Virginia Commonwealth University School of Medicine, Medical College of Virginia Campus, PO Box 980297, Richmond, Virginia 23298-0297. e-mail: [email protected]; fax: (804) 828-9749. Supported by National Institutes of Health grants R01 CA 83650, RO1 CA 39225 (to A.E.S.), R01 CA 42715, and P20 RR 17695 (to D.C.H.).