Overexpression of p53 protein is not directly related to hepatitis B x protein expression and is associated with neoplastic progression in hepatocellular carcinomas rather than hepatic preneoplasia

Mutation Research 462 Ž2000. 365–380 www.elsevier.comrlocaterreviewsmr Community address: www.elsevier.comrlocatermutres

Overexpression of p53 protein is not directly related to hepatitis B x protein expression and is associated with neoplastic progression in hepatocellular carcinomas rather than hepatic preneoplasia Qin Su

a,b

c , Claus H. Schroder , Gerd Otto d , Peter Bannasch ¨

a,)

a

c

DiÕision of Cell Pathology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany b Department of Pathology, Tangdu Hospital, The Fourth Military Medical UniÕersity, Xi’an, 710038, China DiÕision of Virus–Host-Interactions, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany d Department of Surgery, UniÕersity of Heidelberg, 69120 Heidelberg, Germany Dedicated to Ruggero Montesano on the occasion of his 60th birthday. Received 22 September 1999; received in revised form 4 October 1999; accepted 4 October 1999

Abstract p53 mutations and binding of p53 to hepatitis B virus ŽHBV. x protein ŽHBx. have been suggested as alternative mechanisms of development of hepatocellular carcinomas ŽHCCs. in man, both processes resulting in intracellular accumulation of the protein which is detectable by immunohistochemical approaches. We have examined p53 expression in 149 explanted human livers, including 39 cases infected with HBV and 35 bearing HCC. p53 was demonstrated immunohistochemically in 51% of HCC samples Ž18r35., localized mainly in fast growing poorly differentiated areas. Accumulation of mutant p53 was verified by immunoprecipitation in most of the positive HCC samples Ž14r15., implying occurrence of p53 mutations. No cells positive for p53 were found in 354 preneoplastic hepatocellular lesions examined. This indicates that p53 mutation is associated with progression, rather than early development, of HCC in the low-aflatoxin B1-exposed region. The intracellular distribution patterns of p53 and HBx were different, with the former within nuclei and the latter confined to cytoplasmic compartment. HBx did not coimmunoprecipitate with p53. These data indicate that p53–HBx binding is infrequent, if it really occurs, in HBV-infected human liver, and that it cannot be a common mechanism of HBV-associated hepatocarcinogenesis. In addition, p53 accumulation was also observed in some parenchymal and ductular Žoval. cells in cirrhotic livers and, more frequently, in fulminant hepatitis, being independent of HBx expression, and seemingly associated with the damage andror regeneration of liver parenchyma, perhaps merely reflecting a cellular stress response. q 2000 Elsevier Science B.V. All rights reserved. Keywords: p53 protein; Hepatitis B virus x protein; Human hepatocarcinogenesis; Immunohistochemistry; Immunoprecipitation

AbbreÕiations: AP, alkaline phosphatase; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; ECL, enhanced chemiluminescence; HBV, hepatitis B virus; HBx, hepatitis B virus x protein; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; LSAB, labeled streptavidin–biotin complex; NBT, nitro blue tetrazolium; PCNA, proliferating cell nuclear antigen ) Corresponding author. Tel.: q49-6221-423201; fax: q49-6221-423222; e-mail: [email protected] 1383-5742r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 4 2 Ž 0 0 . 0 0 0 2 6 - 0

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1. Introduction The nuclear protein encoded by p53 tumor suppressor gene participates in cellular functions associated with arrest of cell cycle in G1 and modulation of DNA repair, which are believed to prevent the uncontrolled cell proliferation characteristic of tumor progression w1,2x. The activity of p53 can be disrupted either by mutations or loss of the gene, or by interactions with other cellular proteins or with viral proteins of several tumor-associated DNA viruses Žw3,4x, for more references see Wang, et al. w5x.. The wild-type p53 has a short half-life, and does not accumulate at levels high enough to be detected immunohistochemically under normal conditions w6x. However, the mutant forms of p53 are more stable, its half-life being greatly extended, thereby allowing its immunohistochemical detection w7x. While some studies showed a good correlation between p53-immunoreactivity and p53 gene mutations in malignant tumors Žw8x, for more references see Calzolari et al. w9x., varying degrees of discordance have been revealed more recently w9–12x. p53-immunoreactivity has been described in various human tumors without detectable p53 mutations, which included astrocytomas w13x, gliablastomas w14x, clorectal adenomas w15x, bone and soft tissue sarcomas w16x, renal cell carcinomas w17x, cancers of lung w10x and head and neck w9,10x, as well as hepatocellular carcinomas ŽHCCs. w8,12x. It was also observed in human w18–20x and murine non-neoplastic cells w18x. It has been described that wild-type p53 complexed with some cellular or viral proteins including hepatitis B virus ŽHBV. x protein ŽHBx. is also detectable w21x. The frequency of p53 mutations is high in HCCs developing in patients usually chronically exposed to both aflatoxin B1 and HBV infection w22–26x, and low in the cases associated with chronic HBV infection alone and other causative factors Žreviewed by Montesano et al. w27x.. HBV infection is known to be a most frequent causative factor of human HCC. HBx, a regulatory protein encoded by the fourth reading frame of HBV, has been reported to be able to bind to wild-type p53 in vitro and in vivo w3,5,21,28x, the functional inactivation of p53 by complex formation being regarded as an alternative mechanism of HCC development in HBV-infected liver w28x. Using a panel of six well-characterized

anti-HBx antibodies, we have recently demonstrated HBx-immunoreactivity in more than a half of HCCs and cirrhotic livers infected with HBV, including preneoplastic focal or nodular lesions and extrafocal parenchyma, even though the reactivity was confined to a small number of cells w29x. In addition, we found the frequent occurrence of foci and nodules of altered hepatocytes w30,31x, whose preneoplastic nature has been well established in several animal models w32x, and has been associated with HCC development in man w31x. In this study, we investigated expression of p53 in neoplastic and preneoplastic lesions of the liver and its relationship with HBx expression to assess its possible role in the development and progression of hepatic neoplasia.

2. Materials and methods 2.1. LiÕer specimens and histology As listed in Table 1, 149 explanted livers, 35 bearing HCCs and two with cholangiocellular carcinomas, were examined for p53 expression. Among them, 39 cases were positive for HBV infection as determined by serologic and immunohistochemical detection of HBV antigens and further confirmed by immunohistochemical demonstration of hepatitis B surface and core antigens as described in previous studies w29,31x, 30 being characterized by cirrhosis and nine by acute Žeight cases. or subacute Žone case. fulminant hepatitis with massive hepatic necrosis. Four donor livers and nine livers bearing spaceoccupying lesions other than HCC, without any indication of HBV or hepatitis C virus ŽHCV. infection, were employed as a reference group. The study protocol was approved by the Ethics Commission of the Medical Faculty of the University of Heidelberg. All livers were removed from the patients within 45 min after clamping of the portal vein, and samples were taken, fixed in Carnoy’s solution, and embedded in paraffin as described previously w30x. Serial sections of 3 mm were prepared for staining with hematoxylin and eosin, treatment with the periodic acid Schiff-reaction, and reaction with antibodies. Parts of the specimens were frozen in isopentane at y1508C and stored in air-tight tubes at y808C.

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Table 1 Frequencies of p53-immunoreactivity in parenchymal and ductular Žoval. cells of different liver diseases as well as in hepatocellular carcinomas Abbreviations: HC, hepatocytes; DC, ductular Žoval. cells. Specimens

Cirrhosis Posthepatitic, HBV Posthepatitic, HCV Posthepatitic, autoimmunity Alcoholic Cryptogenic Biliary Other known associationsa Noncirrhotic livers with HCC b Primary sclerosing cholangitis c Metabolic and inborn diseasesd ŽSub.acute massive necrosise Reference livers f

Case number

Cases with HCC Ž%.

113 30 22 3 31 10 13 4 4 8 8 16 13

31 Ž27.4. 18 Ž60.0. 8 Ž36.4. 0 5 Ž16.1. 0 0 0 4 Ž100. 0 0 0 0

p53-immunoreactivity in HC Ž%.

DC Ž%.

HCC Ž%.

25 Ž22.1. 4 Ž13.3. 6 Ž27.3. 0 7 Ž22.6. 2 Ž20.0. 6 Ž56.2. 0 0 2 Ž25.0. 4 Ž50.0. 13 Ž81.3. 0

31 Ž27.4. 7 Ž23.3. 11 Ž50.0. 0 8 Ž25.8. 2 Ž20.0. 1 Ž7.7. 2 Ž50.0. 0 3 Ž37.5. 2 Ž25.0. 12 Ž75.0. 0

15 Ž48.4. 7 Ž38.8. 5 Ž62.5. 3 Ž60.0.

3 Ž75.0.

a

Two cases with genetic haemochromatosis and two with Budd-Chiari’s syndrome. Two cases being HCCs of fibrolamellar type. c Four cases with cirrhosis, four with fibrosis and two of them bearing cholangiocellular carcinomas. d Two cases with Byler’s disease, two with Wilson’s disease, one with oxalosis, one with cystinosis, one with a-antitrypsin deficiency and one with hepatic amyloidosis. e Nine cases associated with HBV infection, three with HCV infection, two with alcoholic abuse and two of unknown etiology. f Four donor livers, five explanted livers, three with polycystic liver disease, one bearing malignant epithelioid hemangioendothelioma and one bearing metastatic carcinoid, and four resected liver specimens, two with focal nodular hyperplasia, one with a metastatic breast carcinoma and one with a metastatic colon carcinoma. b

Histologic diagnosis of the specimens was made by two pathologists ŽQ.S. and P.B.. independently. Preneoplastic foci and nodules of altered hepatocytes, mainly of glycogenotic clear cell and mixed cell types, as well as large- and small-cell changes of the liver parenchyma were identified and evaluated as described previously w31x. HCCs were diagnosed histologically in line with the classification of the World Health Organization w33x and graded from grades I to IV according to Edmondson and Steiner w34x. 2.2. Antibodies Rabbit polyclonal CM1 ŽNovacastra, Newcastle, England. and mouse monoclonal DO-7 Žrecognizing amino acid residues 20–25; Dako, Copenhagen, Denmark. immunoreactive with both wild-type and mutant p53 and relatively insensitive to fixation conditions were mainly used for immunohistochemistry. Mouse monoclonal PAb1801 ŽOncogene Science,

Cambridge, MA. and PAb240 ŽOncogene Science. were generally applied for p53 immunoprecipitaion. The former recognizes an epitope corresponding to amino acid residues 46–55 shared by both wild-type and mutant forms of human p53 under non-denaturation condition, therefore, being able to immunoprecipitate both forms of p53. The latter specifically reacts with a common conformational epitope of p53 Žamino acid residues 213–217. exposed by mutations or denaturation w35,36x. The relative specificity of PAb240 has been used to identify mutant p53 by immunoprecipitation from human HCCs w37x and non-neoplastic cells w19x. A panel of six antibodies was used to detect HBx: rabbit antiserum 36985; rabbit antiserum 98616 Ždonated by Dr. Mark A. Feitelson, Philadelphia, USA., purified previously by cytokeratin 18-immunoabsorption Ž98616FA.; rabbit antisera 70646 and 70606 Ždonated by Dr. HansJurgen Schlicht, Ulm, Germany.; antiserum a-pX ¨ Ždonated by Dr. Stephan Urban, Heidelberg, Germany.; mouse monoclonal antibodies HBxAb-A, B,

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C, D and E, with different recognition sites Žfor references see Su et al. w29x.. Mouse monoclonal antibodies PC10 against the proliferating cell nuclear antigen ŽPCNA; Dako., 3E7 against HBV surface antigen ŽDako., Ks18.04 against cytokeratin 18 ŽProgen, Heidelberg, Germany., rabbit anti-HBV core antigen ŽDako. and anti-Ki-67 antigen ŽDako. were also applied. 2.3. Immunoprecipitation and Western blot detection for p53 Frozen tissues from 15 HCCs and 10 livers with acute fulminant hepatitis, showing immunohisto-

chemical p53-reactivity, were used for p53-immunoprecipitation. Samples from one p53-negative HCC, as well as one donor liver, were used as references. For immunoprecipitation from HCC tissues, differentiation grades of the samples were evaluated with hematoxylin and eosin-stained cryostat sections before homogenization. Liver lysates were prepared by grinding the tissues Ž0.5–1 g for each specimen. in liquid nitrogen and homogenizing them in five volumes of precooled lysis buffer w50 mM Tris–HCl ŽpH 8.0., 120 mM NaCl, 5 mM dithiothreitol, 0.5% Nonidet P-40x containing 2 mM phenylmethylsulfonyl fluoride, 1 mgrml aprotinin, 1 mgrml pepstatin A and 1 mgrml leupeptins. Protein was determined

Fig. 1. Serial sections through HCC immunostained for HBx ŽA. and p53 ŽB.. Wile HBx is negative ŽA., nuclear p53 is evident in a serial section in nearly all tumor cells in the grade III area Župper half in B, high magnification showing the area denoted by larger arrows in A. and in few cells Žsmaller arrow. in the surrounding grade II area Žlower half in B.. AP-LSAB immunostaining, nuclei visualized by counterstaining with hematoxylin: ŽA. 190 = ; C, 380 = .

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by the method of Heinzel w38x. The lysates, each containing 10 mg of protein, were precleared with

369

protein G-sepharose CL-6B ŽGammaBind w plus sepharose w , Pharmacia, Uppsala, Sweden. for 1 h and

Fig. 2. p53 accumulation in parenchymal cells of liver with cirrhosis caused by chronic HBV infection ŽA–D., alcoholic abuse ŽE. or Wilson’s disease ŽF., bearing ŽA–D. or free of ŽE and F. HCC. A and B, p53-immunoreactivity is evident in some hepatocytes Žarrows. scattered within the extrafocal parenchyma ŽA. or beside the fibrotic septum, as well as in some small polygonal liver cells Žarrowheads in B.. ŽC and D. Serial sections immunostained with anti-HBx 70646 ŽC. or anti-p53 DO-7 ŽD., showing two nuclei in a single hepatocyte Žshorter arrow. negative for HBx ŽC. and positive for p53 ŽD.. Longer arrow, bile duct. ŽE and F. p53 accumulation in a small polygonal liver cell Žarrow. near a portal tract ŽPT. in ŽE. and a few hepatocytes Žarrows. in ŽF.. AP-LSAB immunostaining, nuclei visualized by counterstaining with hematoxylin: ŽA. 130 = ; ŽB, C, E, F,. 260 = ; ŽD. 380 = .

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centrifuged to remove the beads. Supernatants from each specimen were incubated with monoclonal PAb1801 Ž4 mg., PAb240 Ž4 mg. or normal mouse immunoglobulin ŽIg. G Ž4 mg; Santa Cruz Biotechnology, Santa Cruz, CA. overnight at 48C. Twenty microliters of protein G-sepharose beads were then added to each tube, followed by incubation for 1 h. The beads were collected by centrifugation, washed three times with the lysis buffer, heated in 2 = sodium dodecyl sulfate ŽSDS. sample buffer at 958C for 5 min, and loaded for SDS-polyacrylamide gel electrophoresis ŽPAGE.. Resolved proteins were electroblotted onto Immobilon-P membranes ŽMillipore, Bedford, MA.. For detection of p53, the blots were dried and incubated with rabbit antibody CM1 diluted 1:6000 for 1 h. After washing with buffer Ž50 mM Tris ŽpH 7.4., 150 mM NaCl. containing 0.05% Tween 20, incubation was with alkaline phosphatase

ŽAP.-labeled anti-rabbit IgG Ž1:2000; Dako.. The reaction was visualized with 5-bromo-4-chloro-3indolyl phosphate ŽBCIP.rnitro blue tetrazolium ŽNBT. solution for about 15 min.

2.4. Coimmunoprecipitation of p53 and HBx Two blots were prepared after immunoprecipitation with PAb1801 as described above, one for p53 detection and the other for HBx detection. For the latter, the reaction was performed with rabbit antibody 70646 and peroxidase-conjugated affinity-isolated anti-rabbit IgG consecutively, and finally visualized through enhanced chemiluminescence ŽECL. reagents according to the manufacturer’s instruction ŽAmersham, Buckinghamshire, England.. To ensure

Fig. 3. p53 accumulation in some ductular epithelial cells Žarrows. in livers with acute fulminant hepatitis associated with HBV infection ŽA–C., with hepatocytes in the residual parenchyma Žlower left. negative for p53 ŽC.. AP-LSAB Immunostaining, nuclei visualized by counterstaining with hematoxylin: ŽA, B. 260 = ; ŽC. 380 = .

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detection of the HBx coimmunoprecipitated with p53, the assay was repeated and the peptide was Table 2 Expression of nuclear p53- and HBx-immunoreactivity in HCCs in livers with and without HBV infection Abbreviations: ALD, alcoholic liver disease; IM, immunohistochemistry; IP, immunoprecipitation; LC, liver cirrhosis; NCL, noncirrhotic liver. Percentages of p53- and HBx-immunoreactive cells were graded into 3q Ž ) 30%., 2q Ž5–30%., q Ž - 5%. and y Žabsence.; immunoprecipitation results were evaluated as positive Žq. or negative Žy.. Samples

Grades

p53-IM a ŽIP b .

HBx-IM c

Associations

90199

II III I II III II III II III II III II II I II I II II III II III II II I II III II III IV II II II II II III I II II III II I II

2q 3q Žq. y 2q 3q q 3q Žq. q Žq. q q q Žq. y y y y y y y y y y y y q 2q 3q Žq. q 3q Žq. 3q Žq. q Žy. q Žq. y y y 3q Žq. y q Žq. y y y y q Žq.

q y 2q 2q q q q q q q q y y y q y y y y y y q q y y y y y y y y y y y y y y y y y y y

LC, HBV

93235

9791 9091 93120 93502 94107 88311 90125 90336 92482 92488 93208 94106 951 964 96136 96203 96279

9637 92254 8939 9639 921091 9689 9742 95178 9553 8932 91712 94112

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Table 2 Žcontinued. Samples Grades p53-IM a ŽIP b . HBx-IM c Associations 92543 967 94115 90335

IV II II d II d

q Žq. q Žq. q Žq. y Žy.

y y y y

NCL, unknown NCL, unknown NCL, unknown NCL, unknown

a

p53-immunoreactivity detected with DO-7. Consistent result obtained by immunoprecipitation using PAb1801 and PAb240 in the HCC samples. c HBx detected by immunohistochemistry mainly using antibodies 70646, HBxAb-D, and B as described previously w29x. d Fibrolamellar carcinoma. b

LC, HBV

probed also with polyclonal antibodies 70606 and a-pX.

LC, HBV

2.5. Immunoprecipitation for HBx

LC, HBV LC, HBV LC; HBV LC, HBV LC, HBV LC, HBV LC, HBV LC, HBV LC, HBV

HCC samples showing immunohistochemical HBx-reactivity, as well as the surrounding cirrhotic liver tissues, from nine HBV-infected patients were used. A donor liver was used as the reference. Liver lysates were prepared from 1 g of tissue for each specimen in five volumes of precooled lysis buffer w20 mM Tris–HCl ŽpH 7.0., 150 mM NaCl, 1 mM EDTA, 1 mM MgCl 2 , 5 mM KCl, 1% Nonidet P-40x containing 2 mM phenylmethylsulfonyl fluoride, 1

LC, HBV LC, HBV LC, HBV LC, HBV LC, HBV LC, HBV LC, HCV

LC, HCV LC, HCV LC, HCV LC, HCV LC, HCV LC, HCV LC, HCV LC, ALD LC, ALD LC, ALD LC, ALD LC, ALD

Fig. 4. p53 accumulation in preneoplastic focal or nodular lesions of glycogen-storing ŽGSF. and mixed cell ŽMCF. types and the neoplastic lesions ŽHCC. of grades I ŽI., II ŽII., III and IV ŽIIIqIV., as evaluated semiquantitatively by percentage of cells immunoreactive for DO-7 Ž3q, ) 30%; 2q, 5–30%; q, - 5%; y, absent.. ns number of lesions examined.

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Q. Su et al.r Mutation Research 462 (2000) 365–380

mgrml aprotinin, 1 mgrml pepstatin A and 1 mgrml leupeptins. Portions of donor liver lysate, containing recombinant HBx of 1000, 100, 10 and 1 ng, respectively, were used for demonstration of sensitivity of the assay. After preclearing with protein A-sepharose CL-4B beads ŽAmersham., the lysates were incubated with a mixture of rabbit polyclonal anti-HBx a-pX and 70606. Normal rabbit Ig ŽDako. at the same concentration was used as a negative control. Immunocomplexes were collected on protein A-sepharose CL-4B beads. For the detection of precipitated HBx a cocktail composed of mouse monoclonal HBxAb-A, B, C, D and E Ž1:30. was applied. Bound antibody was recognized by incubation with AP-labeled affinity-isolated anti-mouse IgG ŽDako. and exposure to BCIPrNBT solution. It was also visualized through incubating the blot with peroxidase-labeled affinity-isolated anti-mouse IgG Ž1:2000; Dako. and ECL reagents as described above. 2.6. Immunohistochemistry For each specimen, sections of at least 5 cm2 total area were examined. p53 was detected mainly with monoclonal DO-7, and the reaction was further confirmed using other antibodies including CM1 and PAb1801. After deparaffinization and rehydration, p53, PCNA and Ki-67 antigen were retrieved by boiling the sections in 10 mM citrate buffer ŽpH 6.0. for 20 min. Serial sections were incubated with a serum-free blocking solution ŽX0909, Dako. for 30 min, and incubated overnight at 48C with anti-p53 DO-7 Ž1:400. or CM1 Ž1:2000., PC10 Ž1:1600. or anti-Ki-67 Ž1:200.. Sections from HBV-infected and other livers were also incubated with rabbit anti-HBx 70646, 70606, 36985 and 98616FA diluted 1:1000– 3000, culture supernatants containing mouse monoclonal HBxAb-B or D diluted 1:20, anti-HBV surface antigen 3E7 diluted 1:300 or anti-HBV core antigen diluted 1:3000. The reactions were visualized using an AP-labeled streptavidin–biotin ŽLSAB. kit

373

ŽDako. and new fuchsin as described previously w30x. The sections were lightly counterstained with Mayers hematoxylin and mounted in Kaiser’s glycerol gelatin ŽE. Merk, Darmstadt, Germany.. Cytokeratin 18-immunoreactivity was demonstrated with the same procedure using mouse monoclonal antibody Ks18.04 diluted 1:150 as a positive control. For negative controls, rabbit and mouse primary antibodies were replaced by normal rabbit Ig and mouse monoclonal antibody control 679.1Mc7, respectively, at the same concentrations. The intensities of reactions were evaluated as strong, moderate, weak or negative. 2.7. Statistical analysis The number of nuclei immunoreactive for p53, demonstrated mainly with DO-7, were evaluated as many Ž3 q , ) 30%., moderate Ž2 q , 5–30%., few Žq, - 5%. or absent Žy, 0%.. In order to investigate the possible association between p53 accumulation and the morphologic changes characteristic of chronic hepatitis and hepatic necrosis, periportal or paraseptal piece-meal and intra-acinar hepatocyte necroses were assessed according to Bianchi and Gudat w39x, and increase of ductular cells Ž) 5 ductules in a portal tract., reflecting ductular proliferation, was evaluated as mild Žq, confined to the portal or septal area., moderate Ž2 q , with periportal or paraseptal parenchyma involved. and pronounced Ž3 q , many ductular cells, with extrafocal parenchyma extensively involved.. The relationship between occurrence of p53-immunoreactivity and early stages of human hepatocarcinogenesis as embodied by foci and nodules of altered hepatocytes and intrafocal small-cell change w31x, was also investigated. Statistical computations were carried out using the software packages S-PLUS, Version 3.4 ŽMathSoft, Cambridge, MA. and StatXact3 for Windows ŽCytel Software, Cambridge, MA.. A multivariate analysis was performed using logistic regression to describe the relationship between p53

Fig. 5. Serial sections through a small clearramphophilic mixed cell focus Žarrows; A and B. and a portion of a large clearramphophilic mixed cell focus Žarrows, C and D. in a HCC-bearing liver with HBV-associated cirrhosis, immunostained for HBx ŽA and C. and p53 ŽB and D. comparatively. HBx is visible in the cytoplasmic compartment of hepatocytes within ŽA. and outside ŽC. the lesions; p53 is negative in the lesions ŽB and D.. AP-LSAB immunostaining, nuclei visualized by counterstaining with hematoxylin: ŽA and B. 300 = ; ŽC and D. 380 = .

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accumulation and 7 other parameters including increase of ductular cells, periportal or paraseptal necrosis and intralobular necrosis of hepatocytes, occurrence of glycogenotic clear cell and mixed cell foci, intrafocal small-cell change and HCC. Cohen’s

Kappa Ž k . and the Spearman’s correlation coefficient Ž r . were applied to evaluate the relationships between p53 and HBx expression and between number of p53-positive cells and PCNA- or Ki-67-labeling indice in HCC and non-neoplastic liver tissues.

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Group differences were analyzed with the Fisher’s exact test and Cochran–Armitage trend test. p - 0.05 was regarded as significant.

3. Results 3.1. Immunohistochemistry for p53 As shown in Table 1, immunohistochemical reactivity for p53 was observed in both neoplastic cells in HCCs Ž18r35, 51.1%; Fig. 1B. and parenchymal andror ductular Žoval. cells in cirrhotic livers ŽFig. 2., irrespective of their association with HCC or HBV infection, as well as in livers with acute or subacute massive parenchymal necrosis ŽFig. 3.. In some cirrhotic specimens, a type of small hepatocyte, known as small polygonal liver cell Ž40., was also positive ŽFig. 2B and E.. The p53-reactivity was localized exclusively within nuclei ŽFigs. 1–3.. In HCCs, the number of cells with p53-positive nuclei varied greatly among the cases, and even different areas of the same specimen ŽFig. 1B.. From the 35 HCCs, a total of 46 tumor portions were examined for the relationship between differentiation grade and p53 expression. The frequencies of nuclear p53 accumulation in the portions diagnosed as grades I, II, III, and IV were 16.7% Ž1r6., 48.1% Ž13r27., 72.7% Ž8r11. and 100% Ž2r2., respectively ŽTable 2, Fig. 4.. The number of p53-positive cells increased with the progression from lower to higher grades ŽTable 2, Figs. 1B and 4., along with PCNAand Ki-67-labeling indices Ž p - 0.05.. In cirrhotic

375

livers, both hepatocytes and ductular Žoval. cells positive for p53 were rare ŽFig. 2., the former being more frequent in HCV-associated cirrhosis Ž p s 0.015.. p53-positive hepatocytes were frequently scattered in paraseptal or periportal areas ŽFig. 2B.. From the 354 preneoplastic foci or nodules composed of altered hepatocytes that were examined, 208 lesions were from HCC-bearing livers and 146 lesions from HCC-free livers with cirrhosis or other chronic disorders. All of these lesions were negative for p53-immunoreactivity ŽFigs. 4 and 5B and D.. p53-immunoreactivity was more frequent in the remaining tissue of livers with massive parenchymal necrosis than in cirrhotic livers, being localized mainly in proliferating ductules ŽFig. 3.. Multivariate analysis revealed that, among the seven morphologic parameters examined Žsee Section 2., intralobular necrosis of liver parenchyma was the only important factor associating with p53 accumulation in both hepatocytes Ž p s 0.01. and ductular Žoval. cells Ž p - 0.01.. 3.2. Lack of histological and subcellular correlation between p53 and HBx HBx was demonstrated in sections of both HCC Ž10r18, 55.6%. and cirrhotic livers Ž16r30, 53.3%. associated with HBV infection, but not in those with acute or subacute fulminant hepatitis Ž0r9.. It was localized in a small number of hepatocytes, within or outside preneoplastic focal lesions ŽFig. 5A and C., as well as in some cancer cells, appearing only in the cytoplasmic compartment. No histological or subcel-

Fig. 6. Immunoprecipitation for p53 using PAb1801 ŽA., for mutant p53 using PAb240 ŽB., and coimmunoprecipitation of p53 and HBx ŽC. with immunohistochemically positive Žp53-IMq . samples from HCCs Žlanes 1–15. and regenerating livers with acute fulminant hepatitis ŽAH, lanes 16–25.. Reference livers ŽRL. including an immunohistochemically p53-negative HCC Ž90335, Lane 26. and a donor liver ŽDL, lane 27. were treated in the same way. As another negative control, lysate from the donor liver was also precipitated with normal mouse IgG to substitute for the anti-p53 ŽDL-, lane 28.. A and B. The immunoprecipitates were separated on 12.5% SDS-PAGE and transferred onto Immobilon-P membranes. Blots were incubated with anti-p53 CM1 and AP-conjugated goat anti-rabbit IgG consecutively, and binding was visualized with NBTrBCIP solution. p53 and its mutant form were found in 14 of the 15 specimens Žwith the p53 signal absent only in lane 8.. The p53 was detected in 6 of the 10 samples of livers with acute fulminant hepatitis Žlanes 16, 17, 20, 23–25 in A., and mutant p53 was precipitated in four of the specimens Žlanes 16, 17, 20 and 25 in B.. Heavy chain ŽIgGH. and light chain ŽIgGL. of mouse IgG were also visualized due to their crossreaction with the goat anti-rabbit IgG. C. Immunocomplexes precipitated by PAb1801 were used for HBx detection after the protein transfer. The blots were incubated with the anti-HBx 70646 and peroxidase-conjugated affinity-isolated anti-rabbit IgG Žcrossreaction with mouse IgG diminished to 5%., and developed through the ECL reaction. HBx was not detected in any of the immunoprecipitates Žlanes 1–26.. Purified recombinant HBx ŽrHBx; 10 ngrlane. was used as a positive control Žlane 27.. Molecular-weight standards ŽMWS; lanes 29 in A and B and 28 in C. were visualized with Coomassie Blue R250, and their positions were shown in kilodaltons ŽkDa. on the right side.

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lular correlation was found between HBx- and p53immunoreactivity in serial sections ŽFigs. 1 and 2C and DFig. 5., although a positive association regarding overall expression was found in HBV-associated HCC Ž p - 0.005.. 3.3. Immunoprecipitation of p53 and HBx Of the 15 HCC specimens showing immunohistochemical p53-reactivity, a positive signal was demonstrated in 14 samples Ž93.3%. by immunoprecipitation using PAb1801 and PAb240 ŽFig. 6A and B, Lanes 1–15.. Of the 10 livers with fulminant hepatitis showing a positive immunohistochemical reaction, p53 was immunoprecipitated in six samples using PAb1801, and in three of them using PAb240 ŽFig. 6A and B, Lanes 16–25.. No signals for p53 were detected in negative controls ŽFig. 6A and B, Lanes 26 and 27.. When immunocomplexes precipitated by PAb1801 were also examined for HBx by application of three different polyclonal antibodies, none of them showed any HBx-specific signals ŽFig. 6C.. The immunoprecipitation with 70606 and a-pX, followed by Western blotting and ECL reaction, demonstrated a definite signal from the donor liver lysate containing 10 ng recombinant HBx, with a strong signal detected in those containing 1000 and 100 ng HBx. However, we failed to detect HBx by the same procedure in any of the samples examined with positive immunohistochemical reactions for HBx Ždata not shown.. This applied to HCC samples Ž n s 9., as well as the surrounding cirrhotic liver tissue Ž n s 9., whether positive or negative for p53.

4. Discussion Mutations of the p53 gene are the most common genetic alterations found in many advanced human malignancies. However, their frequencies in HCCs of different geographic origins vary from 10% to 60% Žreviewed by Tabor w41x.. Point mutations, frequently Ž52%. G to T transversions at codon 249 of exon 7, are detected in more than 50% of cases associated with long-term aflatoxin B1 exposure, this being considered mechanistically related to HCC development in populations Že.g., in Qidong, Guangxi

and Tongan, China and South Africa. exposed to the fungal toxin Žw22–26x, for more references see Montesano et al. w27x.. In contrast, p53 mutations, often at positions other than codon 249, are present in only 10–29% of HCCs associated with other causative factors including chronic infections with HBV or HCV and alcoholic liver disease w24,27,42–47x. In the present study, p53 was detected mainly by immunohistochemistry using representative specimens covering a total area of at least 5 cm2 and including cancer tissues of different grades, the results being confirmed by immunoprecipitation in most specimens. The percentage of p53-positive HCCs occurring in cirrhotic livers was higher in this series Ž48.4%. than in those reported previously from lowaflatoxin B 1 exposure regions Ž10–33%. w11,42– 46,48,49x, and similar to that found in Hungary Ž44%. w50x. While results obtained with immunohistochemistry and immunoprecipitation were consistent for most of the HCC specimens examined Ž14r15., neither wild-type nor mutant p53 was precipitated in 1 HCC sample in which rare hepatocytes positive for p53 were demonstrated by immunohistochemistry Žsee Table 2.. Thus, the occurrence of nuclear reactivity for p53 in less than 5% of tumor cells does not necessarily mean that a p53 mutation has occurred, and should be evaluated with caution. HCCs frequently progress from well Žgrade I. through moderate Žgrade II. to poorly differentiated Žgrades III and IV. forms with increasing tumor size and growth speed, as well as acquisition of the ability to invade. Since percentages of p53-positive HCC specimens of grades I, II, III and IV were 16.7%, 48.1%, 72.7% and 100%, respectively, our data provide further support for the notion that a p53 mutation is a late event in HCC formation w46,49,51–56x. Furthermore, the accumulation of mutant p53 does not necessarily mean that the p53 mutation is a causative factor for the progression of HCC. It may also be a consequence of the progression, merely reflecting increased instability of this gene and other gene loci, as p53 gene is one of the most fragile genes in the cellular genome under certain conditions w57,58x. p53-immunoreactivity has been described in hepatocytes in cirrhotic human livers w49,59–61x, as well as in some ductal and ductular Žoval. cells in the

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livers of rats treated with chemical carcinogens w62x. The accumulation of the nuclear protein, believed to be of wild-type, in these non-neoplastic cells has also been suggested to be associated with the neoplastic transformation w59–61x. The present study demonstrated p53-immunoreactivity in both extrafocal parenchymal and ductular Žoval. cells in cirrhotic livers with high risk of HCC development, due to HBV or HCV infection, or alcohol abuse, as well as in the livers with cirrhosis and advanced fibrosis where risk is relatively low, including primary biliary cirrhosis and primary sclerosing cholangitis. Surprisingly, p53 was observed immunohistochemically in some ductular Žoval. cells and hepatocytes in the majority of specimens with acute or subacute fulminant hepatitis, and was also demonstrated by immunoprecipitation in six of the 10 cases examined. Positive signals were found in four of the six livers by immunoprecipitation using PAb240, indicating the presence of mutant p53. However, no p53-immunoreactive cells were observed in the 354 preneoplastic focal lesions examined, p53 accumulation in extrafocal liver cells being associated neither with the development and progression of the focal lesions nor with the occurrence of HCC, indicating that p53 mutation does not initiate HCC development in our low-aflatoxin B 1-exposure region. Clearly, p53 cannot be used as a biomarker for the putative precursors of HCC. p53 accumulation may in fact be a stress-associated adaptive response of hepatocytes responsible for monitoring DNA repair and regulating the cell cycle, in consideration of its close relationship with loss of liver parenchyma and resulting elevation of hepatocyte proliferation in the nonneoplastic livers. The mechanism of p53 accumulation in nonneoplastic cells is a matter of debate. Accumulation of wild-type p53 in liver cells may reflect stabilization of the protein, as pointed out by several authors after careful studies of the p53 accumulation induced in cultured cells by DNA-damaging agents w18x or transfection of DNA w63x. p53 complexed with heatshock protein 70 or HBx is reported to be greatly stabilized w21,64x. Recently, we demonstrated heatshock protein 70 in some oncocytes and amphophilic hepatocytes in cirrhotic human livers, with the immunoreactivity confined to their cytoplasm w65x. However, we also found that the distribution patterns

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of the protein and p53 were different Žunpublished data., providing no support for direct association between them in vivo. Data from several groups strongly indicate in vitro and in vivo binding between p53 and x protein of hepadnaviruses w3– 5,21,28x, this event being suggested to play an important role in HBV-associated hepatocarcinogenesis w28,66x. However, the interaction between the two proteins was questioned by Henkler et al. w37x in an immunoprecipitation study, in which mutant p53 was detected in 29 of the 31 HCC specimens from Chinese patients chronically infected by HBV. Problems in specificity and conjugation capacity have been demonstrated recently for most of five polyclonal and six monoclonal anti-HBx antibodies collected from five laboratories, these being considered main reasons for the controversy w29x. In the present study, HBx was detected in the cytoplasm of cancer cells and parenchymal cells of some cirrhotic livers infected by HBV, which is in accordance with the previous data from our w29x and several other groups Žw67–69x, for more references see Su et al. w29x.. However, we failed to immunoprecipitate HBx even in the immunohistochemically positive specimens, indicating that the concentration of this peptide was below 10 ngrg of liver tissue. This may reflect the very uneven distribution of the immunoreactivity, only a small number of cells being positive. A remarkable loss of immunoreactivity for HBx and possible complexes between HBx and p53 was avoided by the application of six different antibodies to HBx which were well characterized and found to be suitable for both immunohistochemistry and Western blotting w29x. Discordance of the histological and subcellular distribution of p53 and HBx in HCCs and HBV-infected livers and the lack of coimmunoprecipitation indicate that p53–HBx binding in human liver is not frequent if it really occurs, and inactivation of p53 by binding to HBx cannot be a common mechanism of HBV-associated hepatocarcinogenesis. While the hypothesis regarding p53– HBx binding and the resulting inactivation of p53 w3,4,28x were not supported by our data, an association was found between their overall expression in HBV-associated HCC. HBx, whose oncogenic potential has been well documented w70,71x and whose expression has been demonstrated to be preferentially maintained in preneoplastic and neoplastic hep-

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atocytes w29,69,72x, may indeed play a role in HCC development by transactivating some growth-regulating cellular genes w40,73x, disturbing DNA repair w74,75x or interfering with the control of apoptosis w76x.

w8x

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Acknowledgements The authors gratefully acknowledge the statistical advice of Dr. Axel Benner and the technical assistance of Ditmar Greulich and Holger Grage. We wish to thank our colleagues from the Surgical Department of the University of Hannover for providing some liver specimens, Drs. Mark A. Feitelson, Stephan Urban and Hans-Jurgen Schlicht for provid¨ ing anti-HBx antibodies, and Dr. Walter J. Hofmann for his essential help in organizing the sampling of human liver explants. We would also like to thank Drs. Doris Mayer and Hans Jorg ¨ Hacker for helpful discussions, Dr. Malcolm Moore for his critical reading of the manuscript and Joachim Hollatz for his expert photographic assistance.

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