help button home button Am J Pathol ASIP WHAT IS IT?
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Crawford, D. R.
Right arrow Articles by Sell, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Crawford, D. R.
Right arrow Articles by Sell, S.
(American Journal of Pathology. 2006;169:223-232.)
© 2006 American Society for Investigative Pathology
DOI: 10.2353/ajpath.2006.051284

Separate Origins of Hepatitis B Virus Surface Antigen-Negative Foci and Hepatocellular Carcinomas in Transgenic HBsAg (alb/psx) Mice

Dana R. Crawford*, Stephanie Ostrowski{dagger}, Dilip Vakharia{dagger}, Zoran Ilic{ddagger} and Stewart Sell{dagger}{ddagger}

From the Center for Immunology and Microbial Disease,* The Albany Medical College; the Wadsworth Center,{dagger} New York State Department of Health; and the Ordway Research Institute,{ddagger} Albany, New York


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
We have examined the development and transgene expression in liver lesions of transgenic mice bearing the hepatitis B surface antigen (HBsAg) gene of hepatitis B virus under the control of the albumin promoter (alb/psx) to study liver regeneration and hepatocellular carcinoma (HCC) associated with hepatitis B virus infection. Storage of the HBsAg in the endoplasmic reticulum precedes loss of liver cells and regenerative hyperplastic nodules that do not express HBsAg. Histological analysis indicated that HBsAg-negative foci and nodules arose from liver progenitor cells in the portal zone and lacked mRNA expression. Genomic DNA from eight of nine HBsAg-negative laser capture-excised liver foci showed loss of part of the alb/psx gene, whereas no loss of the actin gene was observed. The alb/psx DNA was intact in adjacent HBsAg-positive tissue. Sequencing of polymerase chain reaction products suggested that alterations in the HBsAg transgene in HBsAg-negative foci occurred via large-scale deletions as opposed to single-site mutations. Southern blot analysis of HCC from 2-year-old transgenic HBsAg mice, however, revealed an intact alb/psx gene. Thus, HBsAg-negative progenitor cells with deletions in the transgene appear to be responsible for compensatory regeneration of the liver, whereas HCCs arise from clonal expansion of hepatocytes with intact alb/psx transgenes.

Hepatocellular carcinoma (HCC) accounts for 1% of new cancers detected per year and more than 2% of cancer deaths in the United States.1 There has been a statistically significant increase in the incidence of HCCs in the United States over the last two decades, from 1.4 in 100,000 to 2.4 in 100,000.2 Because of difficulty in diagnosing early HCC, it has a high mortality rate compared with most other cancers.1 HCC is the predominant cause of cancer mortality in sub-Saharan Africa and southern China.3-6 It causes 65 to 75% of all cancer deaths in males and 30 to 55% of cancer deaths in females in Mozambique and in some provinces of southern China.6-16 Epidemiological studies have identified infection with hepatitis B virus (HBV) and contamination of peanuts or grain with aflatoxin (AFB1) as major and possibly synergistic risk factors in these areas.4-17 Individuals with chronic HBV infection have a 200-fold greater risk of developing HCC than do age-matched noninfected controls.1

Multiple factors appear to contribute to HBV infection-induced pathogenesis of liver cancer. Chronic infection and production of cytokines play roles in the development of fibrosis and in liver cell proliferation.15-21 Disruption or promotion of genes associated with the cell cycle, growth, and oncogenic pathways that are present in close proximity to the site of HBV integration have been implicated in transformation and cancer development.21-26 Similarly, HBV-encoded proteins can contribute to the pathology of the cells of the liver.21,27-31

Various aspects of HBV biology and molecular pathogenesis can be addressed through the development of transgenic mouse models with organ-specific expression of viral genes. In the transgenic mouse lineage 50-4 or Tg(Alb-1HBV)Bri44 developed by Chisari et al,32 the HBV BamHI/BglII gene fragment is linked to the albumin promoter directing expression in liver cells. This hepatitis B surface antigen (HBsAg) transgenic mouse lineage with a C57BL/6 background expresses the HBsAg polypeptide in the cytoplasm of hepatocytes but does not express the X-protein.33 The hepatocytes of the mice of this line do not secrete the large HBsAg but rather store it in the endoplasmic reticulum, resulting in liver dysplasia at about 3 months of age.34 This is followed by severe prolonged hepatocellular injury and hepatocyte death, increased generation of reactive oxygen species, regenerative hyperplasia beginning at about 6 months of age, and aneuploidy, oval-cell proliferation, nodule formation, and eventual development of HCC at 15 to 18 months of age in males.32-38 One of the characteristic observations in these mice has been the progressive loss of HBV-specific antigen in the liver corresponding to the appearance of HBsAg-negative microscopic nodules. Later, HCCs develop, and these are also HBsAg negative.33,34 Thus, there appears to be a relationship between HBsAg nodular regeneration and HCC development.

Southern blot analysis of genomic DNA probed with full-length HBV DNA probe showed identical HBV-specific fragments in HCC from all of the 16 mice examined as well as in the tail samples from these mice.33 The authors suggested that despite the apparent integrity of the integrated transgene, the cells of HCCs are transcriptionally altered. Here, we use immunohistological analysis of HBsAg expression to study the cellular origins of HBsAg-negative foci and nodules. In addition, we examine the expression and gene structure of HBsAg in premalignant liver foci as a means to understand the loss of HBsAg protein expression, a step believed to precede development of HCC in this model.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Transgenic HBsAg Mice

Transgenic HBsAg mice were obtained from Dr. Francis Chisari.32,33 This transgenic strain contains an HBV BglIIa DNA fragment coding for the entire HBsAg antigen under the transcriptional control of the mouse albumin gene promoter directing expression to liver cells (alb/psx). The inserted alb/psx gene segment also includes sequences coding for the X-protein of HBV, but the X-protein is not expressed in this transgenic lineage. The official designation for these mice is Tg(Alb-1HBV)Bri44, and they are more commonly referred to as lineage 50-4.33,35 These mice were housed and maintained at the Wadsworth Center Animal Facility under institutionally approved conditions.

Immunohistochemistry

Tissue blocks from the liver were fixed in buffered formalin for 8 hours, embedded in paraffin, and cut into 6-µm-thick sections according to previously described conditions.35,38 Sections were deparaffinized and rehydrated using standard methods including xylene and decreasing concentrations of ethanol. Before staining, antigen retrieval was performed using 0.1% trypsin in 0.05 mol/L Tris-HCl, pH 7.8, and 0.1% CaCl2 buffer at 37°C for 20 minutes. After antigen retrieval, sections were rinsed in water for a total of 10 minutes and incubated in a 1:50 dilution of 30% H2O2 in 100% methanol. Sections were then rinsed in water for a total of 10 minutes and equilibrated in 1x Tris-buffered saline.

Double labeling for HBsAg and cytokeratin was performed sequentially. HBsAg was labeled using goat anti-HBsAg monoclonal antibody (1:1000 dilution; Dako-cytomation, Carpinteria, CA) followed by horseradish peroxidase-labeled donkey anti-goat IgG (1:500; Jackson Immunoresearch, West Grove, PA). Cytokeratin labeling of bile ducts was performed as described previously39 using rabbit anti-cytokeratin wide spectrum (1:100; Dakocytomation) and alkaline phosphatase-labeled anti-rabbit IgG (1:500; Santa Cruz Biotechnology, Santa Cruz, CA). Pan-cytokeratin (Pan-CK) labeling was developed using NBT/BCIP, and HBV staining was developed using NovaRed (Vector Laboratories, Burlingame, CA).

In Situ Hybridization

To determine the expression of the HBsAg in the nodules, we embedded liver tissue from 9-month-old HBV-transgenic mice in OCT compound (Sakura Finetek, Inc., Torrance, CA) and kept it frozen at –80°C until cryo-dissection. In situ hybridization for HBsAg mRNA in liver sections was performed using digoxigenin-labeled RNA probes prepared in both the sense and antisense direction according to the instructions in the DIG RNA Labeling kit supplied by to the manufacturer (Roche, Indianapolis, IN). The DNA template for HBsAg was plasmid pRc/CMV-HBs(S) containing a 2-kb BssHII-KpnI fragment of HBs(S).

Laser Capture Microscopy

Laser-assisted microdissection was performed on formalin-fixed, paraffin-embedded 6- to 8-µm liver sections using the PALM Robot-Microbeam laser microdissection system (PALM Microlaser Technolgies, GmbH, Bernried, Germany). The system is based on a sharply focused laser beam used to cut a circle (approximately 300 to 600 µm in diameter) around a selected area of cells. Sections of liver nodules negative for HBsAg and extra-nodular areas positive for HBsAg were laser-dissected and collected separately into 0.5-ml polymerase chain reaction (PCR) tubes containing 30 µl of catapult/digestion buffer as instructed by PALM Microlaser Technologies (1 µmol/L ethylenediamine tetraacetic acid [pH 8.0], 20 µmol/L Tris [pH 8.0], 0.5% Igepal CA-630 [Sigma, St. Louis, MO], and 0.2 µg of proteinase K). Samples were digested at 55°C for 18 hours and then heat-inactivated for 5 minutes at 95°C. Aliquots of these were used in the PCR reaction mixture together with the appropriate PCR primers as described below.

PCR

Three sets of overlapping genomic PCR primers and corresponding nested primers obtained from Integrated DNA Technologies, Inc. (Coralville, IA) were used to amplify the entire length of the HBsAg transgene (see Results for further description of probes). The microdissected tissue samples obtained as described above from HBsAg-negative or -positive pools were tested separately in the PCR reaction with PCR primers for ß-actin and three sets (I, II, and III) of HBV genomic primers, followed by reamplification of 2 µl of PCR-reaction mixture product with HBV-nested primers. PCR was performed in a Perkin Elmer 480 Thermal Cycler (Perkin Elmer, Boston, MA) for 45 cycles with successive denaturation, annealing, and extension stages set at 95, 55, and 72°C, respectively. The conditions for the two PCR reactions were identical.

Southern Blot Analysis

Fresh tissues obtained from visible liver tumors, kidney tissue, and nontumor liver tissues from 2-year-old transgenic mice were processed for DNA extraction using the GenElute Mammalian Genomic DNA Miniprep kit (Sigma). Approximately 20 µg of DNA was digested with EcoRI restriction enzyme, and DNA fragments were separated on 1% gel agarose and transferred to Gene Screen membrane (NEN Life Sciences, Boston, MA). These blots were then probed with PCR products labeled with Megaprime DNA (Amersham Biosciences, Piscataway, NJ) from HBV-gene template using a standard technique as described.40,41 The primer set II product described above was used as a template for labeling HBV.

Sequencing

After electrophoresis, PCR product amplicons were excised and DNA purified using the Roche gel excision kit according to the manufacturer’s instructions. Extracted DNA was then cycle sequenced by the Wadsworth Center Molecular Genetics Core Facility under the direction of Dr. Matthew Shudt.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Generation of HBsAg-Negative Foci and Nodules in Transgenic HBsAg Mice

The sequence of pathological changes in HBsAg transgenic mice is as follows: 1) storage of HBsAg in the endoplasmic reticulum, leading to large swollen hepatocytes beginning at birth; 2) hepatocyte injury and dysplasia beginning at about 3 months; 3) cell death, development of hyperplastic nodules, and oval cell proliferation, beginning about 6 months; and 4) hepatocellular carcinoma beginning about 15 months in males and death by 18 months. A similar sequence occurs in females, but in many animals there is regeneration of the liver with HBsAg negative cells and survival for over 2 years without development of HCC.38,42 For the present study, we targeted events occurring after the initiation of injury and regenerative hyperplasia in males but before the formation of visible HCC. Liver sections from HBV transgenic mice at various ages were prepared and analyzed for the expression of HBsAg protein. At 5 months of age, essentially all hepatocytes of transgenic HBsAg mice contain HBsAg, but other than enlarged hepatocytes, the structure of the liver is not different from that of nontransgenic mice (Figure 1, A and B) . By 7 to 8 months, foci of HBsAg-negative cells are seen (Figure 1C) . There is a progressive increase in cells not expressing HBsAg, first in small foci, then in nodules (Figure 1, D and E ; 12 months), and finally in multiple nodules and microscopic HCCs (Figure 1F ; 15 months). Essentially all HCCs in this model are HBsAg negative.34,35 Oval cell proliferation is seen after 8 to 10 months (not shown). These results are consistent with previously published reports and suggest that HBsAg-negative nodules arise from HBsAg-negative foci and that HCCs arise from HBsAg-negative nodules.34,35,43 Sections showing early HBsAg foci and nodules, such as that shown in Figure 1, C–E , suggest that early foci are closely associated with ducts in the portal zone.


Figure 1
View larger version (145K):
[in this window]
[in a new window]
  		Figure 1. A–F: HBsAg staining. A, normal liver; B–F, transgenic male mice (brown color). G–I, Pan-CK staining (black color). A, normal mouse liver, 15 months; original magnification, x400; B–F, transgenic HBsAg mice. B, 5 months; original magnification, x400; C, 7 months; D; original magnification, x200; E; original magnification, x400; F, 15 months; original magnification, x20. B: There is uniform accumulation of HBsAg and dysplasia in each hepatocyte at 5 months. C–E: By 7 months, foci of small HBsAg-negative hepatocytes next to ductules (arrow) appear. An HBsAg-negative microcarcinoma is shown in F next to an HBsAg nodule at 15 months. G, Normal male mouse, 9 months; original magnification, x200; H, transgenic male, 2.5 months; original magnification, x200; I, transgenic male, 9 months. Arrows in I point to Pan-CK-positive small ductules extending into hepatic plates. p, portal vein.

 
To clarify a possible relationship between early foci and ductal proliferation, we stained sections with antibodies to cytokeratin to reveal possible duct formation as part of the process of HBsAg focus formation. Figure 1, G–I , shows examples of this staining in normal mice and transgenic mice at 2.5 months and in transgenic mice at 9 months. At 2.5 months (Figure 1H) , small ducts were limited to the portal zone in both normal nontransgenic mice (not shown) and in transgenic mice. By 9 months, there was a progressive increase in small ducts extending from the portal zone into the adjacent hepatic lobules in transgenic mice (Figure 1I) , but the location of ducts in nontransgenic mice was the same as seen at 2.5 months (Figure 1G) . To show the location of HBsAg foci, we cut 20 serial sections to trace the location of the foci in the liver. As shown in Figure 2 , top panels, the HBsAg-negative focus in 9-month-old male transgenic mice can be followed from the liver lobule to the portal zone and then back to the lobule. This suggested that the HBsAg-negative foci have a close relationship with the portal zone and to biliary ductules. To examine the relationships between these ducts and HBsAg nodules more closely, we double-stained sections for Pan-CK and for HBsAg. Twenty serial sections were cut from each of five transgenic HBsAg mice at 8 months of age. Figure 2 , middle panels, shows representative selected serial sections from one of these mice. The arrows in the middle panels point to a close association of the small HBsAg focus with Pan-CK-positive small ducts. A total of 76 HBsAg-negative foci were followed in this manner, and 78% of these could be shown to be adjacent to small biliary ductules. We chose livers from 8-month-old male mice for further analysis of HBsAg expression because at this age, especially in male mice, consistent and well-defined HBsAg nodules could be obtained.


Figure 2
View larger version (111K):
[in this window]
[in a new window]
  		Figure 2. Sequential sections of HBsAg-negative foci. Top panel: HBsAg staining, 8-month-old transgenic male mouse (original magnification, x200). Middle panel: Double staining for HBsAg (red) and Pan-CK (blue), 8-month-old transgenic male mouse. In the top panel, an HBsAg-negative focus can be followed from the liver lobule to the portal zone and then back to the lobule. In the middle panel are representative sections from 20 serial sections. The arrows point to small bile ductules associated with an HBsAg-negative focus. p, portal vein; n, HBsAg-negative focus. Bottom panels: mRNA expression in the liver foci of transgenic mice. Liver sections from 7-month-old HBV transgenic mice analyzed for HBsAg mRNA using in situ hybridization. A, mRNA (Green); B, nuclei (red, propidium iodide); C, double stained. Lack of HBsAg mRNA is observed in foci compared with surrounding (nonfocal) tissue.

 
HBsAg mRNA Expression Is Absent in the Liver Foci of Transgenic Mice

To determine whether the observed absence of HBsAg protein expression was due to a loss of protein antigenicity, to a loss of translatable mRNA, or to a loss of total mRNA, we analyzed liver sections from 9-month-old HBV transgenic mice for the expression of HBsAg mRNA. This was performed by in situ hybridization using the complete HBsAg-region DNA as a probe. HBsAg mRNA expression was not observed in foci (Figure 2 , bottom panels). Thus, the loss of HBsAg protein expression in liver foci is due to the loss of mRNA template.

Analysis of HBsAg Genomic DNA in Liver Foci

The loss of HBsAg mRNA expression suggested the possibility that chromosomal changes in the HBsAg transgene occurred, in turn preventing HBsAg transgene transcription. To address this possibility, we analyzed the genomic DNA in HBsAg-negative foci. Laser capture microscopy was used to obtain small areas of tissue situated completely within foci, and the transgene was analyzed in these areas using PCR. The laser capture approach was important for two reasons. First, it ensured that tissue from one focus at a time was isolated. This avoids the potential heterogeneity that may occur in less accurate focus removal, during which combining of foci may occur. Second, the surrounding (nonfocal) tissue in which HBsAg mRNA expression was evident could be excluded. An approximately 300 to 600 µm diameter area could be routinely cut from a focus, clearly avoiding surrounding HBsAg-positive tissue.

We used PCR analysis of DNA extracted from individual foci to determine whether there was genomic loss of the HBsAg transgene. Genomic DNA was prepared as described in Materials and Methods. Several PCR primers combinations were chosen to generate overlapping amplicon product within the HBsAg transgene, as shown in Figure 3A .


Figure 3
View larger version (78K):
[in this window]
[in a new window]
  		Figure 3. PCR analysis. A: PCR primers used for amplification of the HBV transgene. Location of three PCR primer combinations with internal nested primer sites in the alb/psx transgene. For clarity, two panels are shown. Top panel: Overlapping PCR primers only. Bottom panel: Same as top panel but with internal nested PCR primer sites indicated. B: Specificity of PCR primers. Genomic DNA from positive control transgenic liver (+) and six nontransgenic mouse livers (S1 to S6) was extracted and PCR amplified using the primers described in the top panel of A (listed to the left of the figure), followed by re-amplification with the nested primers shown in the bottom panel of A. Lane 1, size markers. Lane 2, positive control (+). Lane 3, PCR performed in the absence of DNA template negative control (–). Lanes S1 to S6, nontransgenic liver samples. C: HBsAg-negative foci. Genomic DNA from positive control transgenic liver (+) and nine immunohistochemically HBsAg-negative individual nodules (lanes F1 to F9, F1 to F3, F4 to F6, and F7 to F9 are from three different mice) was extracted and PCR amplified using the primers described in the top panels of A and then with the nested primers shown in the bottom panel of A (listed on the left). Lane 1, size markers. Lane 2, positive control (+). Lane 3, negative control (–). Lanes F1 to F9, HBsAg-negative foci samples. D: HBsAg-positive tissue surrounding the HBsAg-negative foci. Genomic DNA from positive control transgenic liver (+) and six samples of immunohistochemically HBsAg-positive cells surrounding the foci (lanes P1 to P6) was extracted and PCR amplified using the primers described above (listed on the left). Lane 1, size markers. Lane 2, positive control (+). Lane 3, negative control (–). Lanes P1 to P6, HBsAg-positive tissue samples. P1 and P2 are from the same mouse as F1 to F3 in C; P3 and P4 are from the same mouse as F4 to F6; and P5 and P6 are from the same mouse as F7 to F9.

 
To assess the specificity of these primers, we analyzed positive control transgenic liver and negative control nontransgenic liver samples. As expected, the transgene-positive control sample generated PCR product with all three overlapping HBsAg primers (Figure 3B , lane +), whereas nontransgenic liver did not show any PCR product with the same primers (Figure 3B , lanes S1–S6). These results confirmed the specificity of the primers and their utility in the analysis of tissue samples.

Genomic DNA from nine immunohistochemically HBsAg-negative foci was then analyzed (Figure 3C) . Detection of PCR product from the ß-actin gene was observed in all foci. However, parallel analysis of the same focal DNA with HBsAg primers produced products with only some of the primer pairs and samples, indicating a loss of DNA. Specifically, three of nine samples exhibited a loss of DNA in the primer set 1 region, five of nine exhibited a loss of DNA in the primer set 2 region, and six of nine exhibited a loss of DNA in the primer set 3 region. Altogether, eight of nine foci lost at least some of the HBsAg gene. In contrast, DNA extracted from six regions surrounding the foci that were immunohistochemically positive for antibody to HBsAg contained DNA template to the complete set of HBsAg primers as evidenced by the production of PCR product in all samples and with all primer sets (Figure 3D) . These results indicate that a probable fragmentation of the HBsAg gene has occurred in the foci but that the same transgene is intact in surrounding tissue that is able to produce HBsAg protein. Thus, altered DNA in the foci is the apparent explanation for the loss of HBsAg mRNA and protein expression in these cells.

Sequencing of Focal Amplicons

All amplicons produced using focal DNA were excised from gels and cycle-sequenced. In every case, an exact match with the HBsAg transgene was observed. These results suggest that alterations in the HBsAg transgene in foci, as evidenced by the absence of product, were due to larger-scale deletions as opposed to single-site mutations.

The HBV Gene in HCC from 2-Year-Old Mice Is Intact

Southern blot analysis was performed using DNA extracted from fresh liver tumors and kidney from 2-year-old transgenic HBsAg mice (Figure 4) . A 5-kb DNA fragment was identified in the EcoRI digest using HBV Set II primer-derived PCR product as a probe. Samples from seven tumors (T1 to T7) obtained from seven separate mice and samples of liver surrounding the tumors (L1 to L3) and a kidney tissue positive control (+) showed the same-sized HBsAg DNA band. The liver sample from normal B6 mouse (–) was negative for the HBsAg gene. Thus, the alb/psx transgene in the HCC from livers of the 2-year-old mice appears to be intact.


Figure 4
View larger version (27K):
[in this window]
[in a new window]
  		Figure 4. Southern blot analysis of 2-year-old HBV transgenic mice liver tumors. T1 to T7, samples from seven different liver tumors from seven different mice; L1 to L3, liver tissue surrounding the tumors: +, HBV transgenic mouse kidney positive control; and –, nontransgenic liver negative control. Approximately 20 µg of extracted genomic DNA was digested with EcoRI, electrophoresed, transferred to Gene Screen, and probed with labeled PCR product (primer set II) to the HBV gene.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The present study suggests a separate cellular origin of regenerative nodules and HCCs in transgenic hepatitis B mice. There is loss of HBV-specific antigen in regenerating foci and nodules in the liver preceding the development of HBsAg-negative HCCs. This observation and other similarities between nodules and HCCs43 support the conclusion that the HBsAg-negative HCCs arise from such negative foci.33-36,43 However, the present studies show that the regenerative nodules show deletions in the inserted alb/psx transgene, whereas HCCs contain the intact transgene, suggesting separate origins for most regenerating nodules and for HCCs.

In their initial studies, Chisari et al33 observed that 50-4 transgenic HBsAg mice exhibited reduced expression of the HBsAg gene with increasing age and that most nodules in these mice lost expression of the HBV gene. Southern blot analysis revealed no difference between the genetic makeup of the DNA in the tumors and that of the tail DNA of the transgenic HBsAg mice33 but a major decrease in the level of HBV mRNA.34 Essentially all of the HCCs that were examined by histological staining did not contain HBsAg.34,35 Thus, despite the apparent integrity of the alb/psx transgene in HCCs, the HBsAg is not expressed in HCCs.

On the other hand, our PCR analysis of immunohistochemically negative areas of the nodules in liver tissue sections from 9-month-old transgenic HBV mice using primers directed against the promoter and pre-S1 region reveal that part of the alb/psx transgene is missing in eight of nine HBsAg-negative nodules. Thus, we conclude that deletion of DNA in the regenerating nodules is the apparent explanation of the loss of HBsAg mRNA and protein expression in most of the regenerative nodules in the transgenic alb/psx mice. It is not clear how this loss of HBV gene occurs or how it correlates with subsequent cancer formation. In most cases, these HBsAg-nonexpressing cells appear to function as normal hepatocytes, and they eventually may replace the cells damaged by storage of HBsAg.38,42 Overall, two types of DNA changes may occur, one giving rise to regenerating nodules (alteration of the transgene) and the other resulting in HCC with no alteration of the transgene. The facts that only one of nine nodules has an intact transgene and that HCCs have intact transgenes may be related to the finding that in this model of hepatocarcinogenesis, as well as in experimental hepatocarcinogenesis, there may be hundreds of nodules in one liver, but only one to three HCCs arise.44 Thus, the nodules with the intact transgene may be the ones that will become HCCs.

The HCCs in these mice are slow growing and do not metastasize unless the mice are exposed to a carcinogen, such as aflatoxin.42,45 Co-carcinogens released from the area of cell death, such as reactive oxygen species,46,47 may also contribute to transformation and growth of the tumor cells, perhaps even contributing to loss of HBsAg expression despite the apparent integrity of the transgene.46-48 During this process, some progenitor cells in the liver most likely develop additional changes leading to HCC, particularly in male mice. The genetic nature of the putative changes in the HCC is not yet known, because neither alterations in the inserted alb/psx transgene nor mutations in specific oncogenes or suppressor genes have been identified in the HCCs arising in these mice.49,50

The involvement of liver progenitor cells in the generation of nodules and HCC is supported by the microscopic location of newly developing lesions. Our present study shows proximity of HBsAg-negative foci and nodules to portal zones, where putative liver stem cells (oval cells) are located.51-53 In addition, microcarcinomas appear to develop adjacent to portal areas. For example, the arrows in Figure 1F point to the border between an HBsAg-negative microcarcinoma (top) and an HBsAg-negative nodule. We performed three-dimensional analysis with a 20-section series to elucidate a possible role of cells within the biliary tree in the formation of the HBsAg-negative foci. We conclude that there is continued regeneration of antigen-negative foci from liver progenitor cells within or adjacent to the biliary system. These cells are stimulated to proliferate because of the loss of mature hepatocytes and decreased ability of mature hepatocytes to proliferate due the HBsAg storage injury. Survival of the newly formed hepatocytes is accomplished by alteration of the alb/psx transgene so that it is not expressed. During this process neoplastic nodules of various grades appear with development after 15 months of surface antigen-negative HCCs. Females living longer than 18 months appear able to regenerate large zones of apparently normal HBsAg-negative liver and to reconstitute their livers with normal appearing cells,38 similar to what occurs in uPA transgenic mice.54,55 We postulate that liver regeneration is accomplished through a continued proliferation of "immature" hepatocytes or tissue-determined stem cells and that these gradually replace the injured surface antigen-expressing hepatocytes.

The observation that foci and nodules arise from the portal zone is consistent with the possibility that these newly generated tissues arise from bone marrow precursors.56-58 However, Vig and co-workers59 did not find bone marrow donor-derived foci or nodules in female 6-month-old transgenic mice that had been irradiated and transplanted with male bone marrow. Similarly, we have not identified bone marrow-derived foci, nodules, or HCCs when the mice female transgenic HBsAg mice reconstituted with male bone marrow are sacrificed at 18 to 22 months of age (I. Guest, Z. Ilic, and S. Sell, unpublished data).

The expression of HBsAg in liver cells and HCC in humans with HBV infection has a completely different mechanism and meaning from that in the transgenic HBV mice. HBsAg was detected in about one-third of HCCs arising in humans who were serologically positive for HBV.60 This contrasts with the lack of expression of HBsAg in essentially 100% of the HCCs in the transgenic alb/psx mice. In humans, HBsAg expression reflects infection of the cells with HBV60,61 ; in the transgenic mice, expression is due to transcriptional activation of the alb/psx transgene.32-36 Thus, expression of the HBsAg reflects infection of the HCC cells with HBV. The mechanism of liver cell damage in humans with HBV infection is killing of infected hepatocytes by cytotoxic T lymphocytes reacting to viral antigens.36 On the other hand, expression of HBsAg in the transgenic HBV mice is directly responsible for death of the transgenic hepatocytes, with little associated inflammation.

A comparison of the pathogenesis of HCC in humans with that in transgenic HBsAg mice also indicates major differences.36,61 The development of HCC in humans is attributed to several different mechanisms, including 1) deregulation of cell growth control genes by viral genes that have integrated into hepatocytes,18-24,62-65 2) oxygen radical-mediated damage to DNA secondary to activated macrophages recruited in response to immune inflammation,18,20,36,61 3) regeneration resulting in proliferation of hepatic precursor cells,41,51,60 and 4) activity of the viral transactivating protein (HBx).18,23,27-31,66-68 Because there is no evidence for production of HBx or for integration of HBV DNA other than the alb/psx transgene in the transgenic HBV mice, the pathogenic pathways in common between the transgenic mice and human HBV-associated HCC are chronic liver injury, oxygen radical production, and proliferation of hepatocytes and liver progenitor cells.33,42,61


   Acknowledgements
 
We thank Drs. Ian Guest and Simon Spivak for assistance and helpful suggestions and Lin Tong for in situ mRNA analysis of foci. We also thank Dr. Francis Chisari for the HBV transgenic mice.


   Footnotes
 
Address reprint requests to Stewart Sell, M.D., Wadsworth Center, New York State Department of Health, Albany, NY 12208. E-mail: ssell{at}wadsworth.org

Supported by National Institutes of Health grant ES009495 (to S.S.).

Accepted for publication March 17, 2006.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. : American Cancer Society: Cancer Facts and Figures, 2004. 2004 American Cancer Society, Inc. Atlanta, GA
  2. El Serag HB, Mason AC: Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med 1999, 340:645-750[Free Full Text]
  3. Ozturk M: p53 mutation in hepatocellular carcinoma after aflatoxin B1 exposure. Lancet 1991, 338:1356-1359[CrossRef][Medline]
  4. Munoz N, Bosch F: Epidemiology of hepatocellular carcinoma. Okuda K Ishak K eds. Neoplasms of the Liver. 1987:pp 13-19 Springer-Verlag, Tokyo
  5. Bressac B, Kew M, Wands J, Ozturk M: Selective G to T mutations of p53 gene in hepatocellular carcinoma from Southern Africa. Nature 1991, 350:429-431[CrossRef][Medline]
  6. Yeh FS, Yu MC, Mo CC, Luo S, Tong MJ, Henderson BE: Hepatitis B virus, aflatoxin and hepatocellular carcinoma in Southern Guangxi, China. Cancer Res 1989, 49:2506-2509[Abstract/Free Full Text]
  7. Van Rensburg SJ, Cook-Mozaffari P, Van Schalkwyk DJ: Hepatocellular carcinoma and dietary aflatoxin in Mozambique and Transkei. Br J Cancer 1985, 5:713-726
  8. Yu SZ: Epidemiology of primary liver cancer. Tang ZY eds. Subclinical Hepatocellular Carcinoma. 1989:pp 189-212 Springer-Verlag, Berlin
  9. Agular F, Harris CC, Sun T, Hollstein B, Cerutti P: Geographic variation of p53 mutational profile in nonmalignant human liver. Science 1994, 264:1317-1319[Abstract/Free Full Text]
  10. Beasley RP, Hwang LY, Lin CC, Chien CS: Hepatocellular carcinoma and hepatitis B virus: a prospective study of 22,707 men in Taiwan. Lancet 1981, 2:1129-1133[CrossRef][Medline]
  11. Delong S: Drinking water and liver cell cancer: an epidemiologic approach to the etiology of this disease in China. Chinese Medical J 1979, 92:748-756
  12. Sun T-T, Chu Y-Y: Carcinogenesis and prevention strategy of liver cancer in areas of prevalence. J Cell Physiol Suppl 1984, 3:39-44[Medline]
  13. Sun T, Wu S, Wu Y, Chu Y: Measurement of individual aflatoxin exposure among people having different risk for primary hepatocellular carcinoma. Hayashi Y Nagao M Sugimura T Takayama S Tomatis L Wattenberg LW Wogan GN eds. Diet, Nutrition and Cancer. Proc. 16 Int Symp. of Princess Takamutsu Cancer Res. Fund 1986:pp 225-235 Japanese Science Society Press, Tokyo
  14. Li KC, Li H-P, Wong S-S, Chuang Y-S: Survey of aflatoxin contaminations in peanuts from various areas in Taiwan. 1977:p 37 Taiwan, Taiwan Agricultural Chemical and Toxin Substances Research Institute, Taichung
  15. Chen C-J, Wang L-Y, Lu S-N, Wu N-H, You S-L, Zhang Y-J, Wang L-W, Santella RM: Elevated aflatoxin exposure and increased risk of hepatocellular carcinoma. Hepatology 1996, 24:38-42[Medline]
  16. Tabor E: Strongly supported features of the association between hepatitis B virus and hepatocellular carcinoma. Tabor E Bisulgi AM Purcell RH eds. Etiology, Pathology, and Treatment of Hepatocellular Carcinoma in North America. 1991:pp 107-117 Portfolio, The Woodlands, TX
  17. Ming L, Thorgeirsson SS, Gail MH, Lu P, Harris C, Wang N, Shao Y, Wu Z, Liu G, Wang X, Sun Z: Dominant role of hepatitis B virus and cofactor role of aflatoxin in hepatocarcinogenesis in Quidong, China. Hepatology 2002, 36:1214-1220[CrossRef][Medline]
  18. Nieters A, Yuan JM, Sun C, Zhang ZQ, Stoehimacher J, Govindarajan S, Yu MC: Effect of cytokine genotypes on the hepatitis B virus-hepatocellular carcinoma association. Cancer 2005, 103:740-748[CrossRef][Medline]
  19. Diao J, Garces R, Richardson CD: X protein of hepatitis B virus modulates cytokine and growth factor related signal transduction pathways during the course of viral infections and hepatocarcinogenesis. Cytokine Growth Factor Rev 2001, 12:189-205[CrossRef][Medline]
  20. Lee MO, Choi YH, Shin EC, Kang HJ, Kim YM, Jeong SY, Seong JK, Yi DY, Cho H, Park JH, Kim SJ: Hepatitis B virus X protein induced expression of interleukin 18 (IL-18): a potential mechanism for liver injury caused by hepatitis B virus (HBV) infection. J Hepatol 2002, 37:380-386[CrossRef][Medline]
  21. Brechot C: Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology 2004, 127:556-561
  22. Levy L, Renard CA, Wei Y, Buendia MA: Genetic alternations and oncogenic pathways in hepatocellular carcinoma. Ann NY Acad Sci 2002, 963:21-36[Abstract/Free Full Text]
  23. Miyaki M, Sato C, Sakai K, Konishi M, Tanaka K, Muraoka M, Kikuchi-Yanoshita R, Nadaoka Y, Kanda H, Kitagawa T: Malignant transformation and EGFR activation of immortalized mouse liver epithelial cells caused by HBV enhancer-X from a human hepatocellular carcinoma. Int J Cancer 2002, 85:518-522
  24. Berasain C, Perara E, Huang SM, Mouly H, Brechot C: Oncogenic activation of a human cyclin A2 targeted to the endoplasmic reticulum upon hepatitis B virus genome insertion. Oncogene 1998, 16:1277-1288[CrossRef][Medline]
  25. Wang J, Chenivesse X, Henglein B, Brechot C: Hepatitis B virus integration in a cyclin A gene in a hepatocellular carcinoma. Nature 1990, 343:555-557[CrossRef][Medline]
  26. De The H, Marchio A, Tiollais P, Dejean A: A novel steroid thyroid hormone receptor-related gene inappropriately expressed in human hepatocellular carcinoma. Nature 1987, 330:667-670[CrossRef][Medline]
  27. Kim WH, Hong F, Jaruga B, Hu Z, Fan S, Liang RJ, Gao B: Additive activation of hepatic NF-kappaB by ethanol and hepatitis B protein X (HBX) or HCV core protein: involvement of TNF-alpha receptor 1-independent and -dependent mechanisms. FASEB J 2001, 15:2551-2553[Abstract/Free Full Text]
  28. Rahmani Z, Huh KW, Lasher R, Siddiqui A: Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential. J Virol 2000, 74:2840-2846[Abstract/Free Full Text]
  29. Chen MT, Billaud JN, Sallberg M, Guidotti LG, Chisari FV, Jones J, Hughes J, Milich DR: A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen. Proc Natl Acad Sci USA 2004, 101:14913-14918[Abstract/Free Full Text]
  30. Lee Yi, Hwang JM, Im JH, Lee YI, Kim NS, Kim DG, Yu DY, Moon HB, Park SK: Human hepatitis B virus-X protein alters mitochondrial function and physiology in human liver cells. J Biol Chem 2004, 279:15460-15471[Abstract/Free Full Text]
  31. Lee MO, Choi YH, Sin EC, Kang HJ, Kim YM, Jeong SY, Seong JK, Yu DY, Dho H, Park JH, Kim SJ: Hepatitis B virus X protein induced expression of interleukin 18 (IL-18): a potential mechanism for liver injury caused by hepatitis B virus (HBV) infection. J Hepatol 2002, 37:380-386[CrossRef][Medline]
  32. Chisari FV, Pinkert CA, Milich DR, Filippi PA: A transgenic mouse model of the chronic hepatitis B surface antigen carrier state. Science 1985, 230:1157-1160[Abstract/Free Full Text]
  33. Chisari FV, Klopchin K, Moriyama T, Pasquinelli C, Dunsford HA, Sell S, Pinkert CA, Brinster RL, Palmiter RD: Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell 1989, 59:145-1156[CrossRef][Medline]
  34. Chisari FV, Filippi P, McLachlan A, Milich DR, Riggs M, Lee S, Palmiter RD, Pinkert CA, Brinster RL: Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice. J Virol 1986, 60:880-887[Abstract/Free Full Text]
  35. Dunsford HA, Sell S, Chisari FV: Hepatocarcinogenesis due to chronic lever cell injury in hepatitis B virus transgenic mice. Cancer Res 1990, 50:3400-3407[Abstract/Free Full Text]
  36. Chisari FV: Hepatitis B virus transgenic mice: insights into the virus and the disease. Hepatology 1995, 22:1316-1325[CrossRef][Medline]
  37. Chisari FV, Filippi P, Buras F, McLachlan A, Popper H, Pinkert CA, Palmiter RD, Brinster RF: Structural and pathological effects of synthesis of hepatitis B virus large envelope polypeptide in transgenic mice. Proc Natl Acad Sci USA 1987, 84:6909-6913[Abstract/Free Full Text]
  38. Ilic Z, Sell S: Dietary cadmium enhances the development of hepatocellular carcinomas in transgenic HBV mice. Carcinogenesis 1994, 15:2057-2060[Abstract/Free Full Text]
  39. Yin L, Lynch D, Ilic Z, Sell S: Proliferation and differentiation of ductular liver precursor cells and non-parenchymal cells during the restitutive response of the rat liver to carbon tetrachloride injury after inhibition of hepatocyte proliferation by N-2-acetylaminofluorine. Histol Histopathol 2002, 17:65-81[Medline]
  40. Abramova NE, Davies KJA, Crawford DR: Polynucleotide degradation during early stage response to oxidative stress is specific to mitochondria. Free Radic Biol Med 2000, 28:281-288[CrossRef][Medline]
  41. Crawford DR, Edbauer-Nechamen CA, Lowry CV, Salmon SL, Kim YK, Davies JMS, Davies KJJA: Assessing gene expression during oxidative stress. Packer L eds. Methods in Enzymology, 1994, vol 234.:pp 175-217 Academic Press, San Diego, CA,
  42. Sell S: Mouse models to study the interaction of risk factors for human liver cancer. Cancer Res 2003, 63:7553-7562[Abstract/Free Full Text]
  43. Toshkov I, Chisari G, Bannasch P: Hepatic preneoplasia in hepatitis B virus transgenic mice. Hepatology 1994, 20:1162-1172[CrossRef][Medline]
  44. Sell S: Distribution of alphafetoprotein and albumin containing cells in the livers of Fischer rats fed four cycles of N-2-fluorenylacetamide. Cancer Res 1978, 38:3107-3113[Abstract/Free Full Text]
  45. Ghebranious N, Sell S: Hepatitis B injury, male gender, alfatoxin, and p53 expression each contribute to hepatocarcinogenesis in transgenic mice. Hepatology 1998, 27:383-391[CrossRef][Medline]
  46. Hagen TM, Huang S, Curnutte J, Fowler P, Martinez V, Wehr CM, Ames BN, Chisari FV: Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma. Proc Natl Acad Sci USA 1994, 91:10771-10778[Abstract/Free Full Text]
  47. Feig DI, Reid TM, Loeb LA: Reactive oxygen species in tumorigenesis. Cancer Res 2004, 54(Suppl 7):1890s-1894s
  48. Uprichard SL, Wieland SF, Althage A, Chisari FV: Transcriptional and post-transcriptional control of hepatitis B virus gene expression. Proc Natl Acad Sci USA 2003, 100:1310-13155[Abstract/Free Full Text]
  49. Pasquinelli C, Bhavani K, Chisari FB: Multiple oncogenes and tumor suppressor genes are structurally and functionally intact during hepatocarcinogenesis in hepatitis B virus transgenic mice. Cancer Res 1999, 52:2823-2829
  50. Wang L, Ilic Z, Sell S: p53, MEM-2 and IGF-II in hepatocellular carcinomas induced by carcinogen exposure of hepatitis B transgenic mice. Transgenics 1995, 1:609-618
  51. Sell S, Leffert HL: An evaluation of cellular lineages in the pathogenesis of experimental hepatocellular carcinoma. Hepatology 1982, 2:77-86[Medline]
  52. Sell S, Salman J: Light- and electron-microscopic autoradiographic analysis of proliferating cells during the early stages of chemical hepatocarcinogenesis in the rat induced by feeding n-2-fluorenylacetamide in a choline deficient diet. Am J Pathol 1984, 114:287-300[Abstract]
  53. Rosenberg D, Ilic Z, Sell S: Proliferation of hepatic lineage cells of normal C57BL and IL-6 knockout mice after cocaine-induced periportal injury. Hepatology 2000, 31:948-955[CrossRef][Medline]
  54. Sandgren EP, Palmiter RD, Heckel JL, Daugherty CC, Brinster RL, Degen JL: Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene. Cell 1991, 66:245-256[CrossRef][Medline]
  55. Braun KM, Thompson AW, Sandgren EP: Hepatic microenvironment affects oval cell localization in albumin-urokinase-type plansminogen activator transgenic mice. Am J Pathol 2003, 162:195-202[Abstract/Free Full Text]
  56. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, Goff JP: bone marrow as a potential source of hepatic oval cells. Science 1999, 14:1168-1170
  57. Theise ND, Badve S, Saxena R, Henegariu O, Sell S, Crawford JM, Krause DS: Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 2000, 31:235-240[CrossRef][Medline]
  58. Sell S: Heterogeneity and plasticity of hepatocyte lineage cells. Hepatology 2001, 33:738-750[CrossRef][Medline]
  59. Vig P, Russo FP, Edwards RJ, Tadrous PJ, Wright NA, Thomas HC, Alison MR, Forbes SJ: The sources of parenchymal regeneration after chronic hepatocellular liver injury in mice. Hepatology 2006, 43:316-324[CrossRef][Medline]
  60. Hsu H-C, Wu T-T, Sheu J-C, Wu C-Y, Chiou T-J, Lee C-S, Chen D-S: Biologic significance of the detection of HBsAg and HBcAg in liver and tumor from 204 HBsAg-positive patients with primary hepatocellular carcinoma. Hepatology 1989, 9:747-750[Medline]
  61. Feitelson MA: Hepatitis B virus infection and primary hepatocellular carcinoma. Clin Microl Rev 1992, 5:275-301
  62. Shafritz DA: Hepatitis B virus DNA molecules in the liver of HBsAg carriers: mechanistic considerations in the pathogenesis of hepatocellular carcinoma. Hepatology 1982, 2:35S-41S
  63. Murakami Y, Saigo K, Takashima H, Minami M, Okanour T, Brechot C, Paterlini-Brechot P: Large scale analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas. Gut 2005, 54:1162-1168[Abstract/Free Full Text]
  64. Tamori A, Yamanishi Y, Kawashima S, Kanehisa M, Enomoto M, Tanaka H, Kubo S, Shiomi S, Nishiguchi S: Alteration of gene expression in human hepatocellular carcinoma with integrated hepatitis B virus DNA. Clin Cancer Res 2005, 11:5821-5826[Abstract/Free Full Text]
  65. Feitelson MA: Parallel epigenetic and genetic changes in the pathogenesis of hepatitis virus-associated hepatocellular carcinoma. Cancer Lett 2005 [Epub ahead of print]
  66. Su Q, Schroder CH, Hofmann WJ, Otto G, Pichlmayr R, Bannasch P: Expression of hepatitis B virus X protein in HBV-infected human livers and hepatocellular carcinomas. Hepatology 1998, 27:1109-1120[CrossRef][Medline]
  67. Feitelson MA, Duan LX: Hepatitis B virus x antigen in the pathogenesis of chronic infections and the development of hepatocellular carcinoma. Am J Pathol 1997, 150:1141-1157[Abstract]
  68. Wang XZ, Chen XC, Chen YX, Zhang LJ, Chen FL, Chen ZX, Chen HY, Tao QM: Overexpression of HBxAg in hepatocellular carcinoma and its relationship with the Fas/FasL system. World J Gastroenterol 2003, 9:2671-2675[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Crawford, D. R.
Right arrow Articles by Sell, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Crawford, D. R.
Right arrow Articles by Sell, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS