Hepatocellular carcinoma (HCC) affects males in a significantly higher proportion than females and is one of the human cancers etiologically related to viral factors. Many studies provide strong evidence of the direct role that hepatitis B virus (HBV) plays in hepatic carcinogenesis, but the functions of HBV surface antigen (HBsAg) and X protein (HBx) in hepatocarcinogenesis through direct or indirect mechanisms are still being debated. We generated two HBV gene knock-in transgenic mouse lines by homologous recombination. HBsAg and HBx genes were integrated into the mouse p21 locus. Both male and female p21-HBx transgenic mice developed HCC after the age of 18 months; however, male p21-HBsAg transgenic mice began to develop HCC 3 months earlier. The expression of a number of genes related to metabolism and genomic instability largely resembled the molecular changes during the development of HCC in humans. ER-β (estrogen receptor-β) was extremely up-regulated only in tumor tissues of male p21-HBsAg mice, providing genetic evidence that HBsAg might be the major risk factor affecting the gender difference in the causes of HCC. In conclusion, these mice might serve as good models for studying the different roles of HBsAg and HBx in early events of HBV-related hepatocarcinogenesis. (HEPATOLOGY 2004;39:318–324.)
Hepatocellular carcinoma (HCC) is one of the most common malignancies and affects males in a significantly higher proportion than females.1 Many risk factors that may cause HCC have been reported, including a diet with a high iron load,2 intake of aflatoxin B1, alcohol-related cirrhosis, and infection with hepatitis virus B or C.3 An abundance of facts has provided proof that HBV infection plays an important role in the development of HCC.
HBV is a DNA virus with a circular, partially double-stranded genome approximately 3.2 kb in length. The genome contains four overlapping open reading frames: surface, core, polymerase, and X.4 Many research studies focus on the X protein because it may function as a transactivator to activate some signaling pathways and transcriptional factors, induce a variety of cellular genes including pro-oncogenes, and interfere with DNA repair and apoptosis.5, 6 Compared with X, the biological function of HBsAg has received less attention. Many types of transgenic mice harboring HBV genes have been reported, including HBsAg and HBx. However, the phenotypes of the transgenic mice generated by the conventional microinjection technique vary considerably with the genetic background due to the random insertion of the HBV genes. Some transgenic mice develop HCC in high proportion,7, 8 while others may not exhibit any hepatic injury.9, 10
To better understand the consequences of the integration of HBV genes, we introduced the HBsAg and HBx genes respectively into the same locus of p21 of the mouse genome by homologous recombination. The HBV gene knock-in transgenic mice expressed either HBsAg or HBx, mainly in the liver tissues, and developed HCC. The differential expression of genes between the livers of wild type and transgenic mice was studied. Some genes that related to metabolism and genomic instability were found up-regulated in tumor tissues of the transgenic mice. Estrogen receptor β (ER-β) was found to be expressed only in the liver tumors from male HBsAg transgenic mice, but not in the ones from female HBsAg transgenic mice and the HBx transgenic mice. This provides new clues to understand the association of HBV genes with the gender difference in HCC.
HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen;HBx, hepatitis B gene X; ER-β, estrogen receptor-β; ES cell, embryonic stem cell; PCR, polymerase chain reaction;neo, neomycin; PCNA, proliferating cell nuclear antigen; ER-α, estrogen receptor-α.
Materials and Methods
To construct a targeting vector for HBsAg, a 2.2-kb BglII-BamHI fragment from pADR-111 was subcloned into pLoxpneo12 to get pLoxpneo-HBsAg. A 2.0-kb XhoI-NotI fragment from p21neo13 that is 5′ to the second exon of the p21 gene and a 6.0-kb XbaI-BamHI fragment from p21neo that is 3′ to the second exon were subcloned into pLoxpneo-HBsAg. To make the targeting construct for HBx gene, a 1.15-kb XbaI-BamHI fragment from pADR-1 and a 0.58-kb BglII-BamHI fragment, also from pADR-1, were subcloned into pLoxpneo successively to get pLoxpneo-HBx. A 2.0-kb XhoI-NotI fragment and a 6.0-kb XbaI-BamHI fragment from p21neo were subcloned into the pLoxpneo-HBx. The finished constructs are shown in Fig. 1A .
Homologous Recombination in Embryonic Stem Cells and Generation of Germline Chimeras.
TC1 embryonic stem (ES) cells were transfected with digested targeting vectors and selected with G418 and FIAU as described previously.14 ES cell clones that were resistant to both G418 and FIAU were picked and analyzed by Southern blotting for homologous recombination events within the p21 locus. Genomic DNAs from these clones and the parental TC1 cell line were digested with EcoRI and BglII, respectively, followed by Southern blots using an 800-bp AccI fragment that is 5′ to the targeting vector (Fig. 1A).
ES cells heterozygous for the targeted insertion were microinjected into C57BL/6 blastocysts to obtain germline transmission. The injected blastocysts were implanted into the uteri of pseudopregnant Swiss Webster (Taconic, Germantown, NY) foster mothers and allowed to develop to give birth. Male chimeras (identified by the presence of agouti coat color) were mated with C57BL/6 and NIH Black Swiss females (Taconic). Germline transmission was confirmed by agouti coat color in the F1 animals, and all agouti offspring were tested for the presence of the insertion by Southern blot analysis using the same conditions as the detection of the homologous recombination event in the ES cells.
Genotypes were determined by Southern blotting or polymerase chain reaction (PCR). For PCR analysis, the wild type p21 allele was detected using primer 1 (5′-TCTTCTGTTTCAGCCA CAGGC-3′) and primer 2 (5′-TGTCAGGCTGGTCTGCCTCC-3′); this primer pair amplifies a fragment of 436 bp from the wild type p21 allele. The primer 3 (5′-ATTTTCCAGGGATCT GACTC-3′), locating 5′ to the insertion, and the primer 4 (5′-CCAGACTGCCTTGGGAAAA G C-3′), locating within the insertion of the neomycin (neo) gene, amplifies a fragment of 215 bp. The primer 5 (5′-GGACCCTGCACCGAACATGG-3') and the primer 6 (5′-GGAATAGCCCCAACGTTTGG-3′), locating within the HBsAg gene, were used to detect the insertion of HBsAg gene. The primer 7 (5′-TCTCTGCCAAGTGTTTGCTGACGC-3′) and the primer 8 (5′-TCGGTCGTTGACATTGCTGAGAGTC-3′), locating within the HBx gene, were used to detect the insertion of the HBx gene.
Reverse Transcription PCR.
Total RNAs were isolated from livers of wild type and transgenic mice using the Trizol method according to the manufacturer's instructions. Reverse transcription PCR was performed by using the mRNA selective PCR kit (Takara, Dalian, China). The primers 5′-GGACGGTGACTCCTACTTCTGT-3′, located in exon 2, and 5′-CCGTGGGC ACTTCAGGGTTT-3′, located in exon 3, which amplify a fragment of 679 bp, were used to detect the transcript of p21; the primers 5′-CACAGGACTAGAACA ACCTGC-3′ and 5′-GCTGGTGAAAAGGACCTCT-3′ were used to detect the transcript of Hprt.
Histology and Immunohistochemistry.
Livers and tumors were fixed in Bouin's solution or formalin solution and embedded in paraffin. Five-micrometer sections were stained with hematoxylin and eosin (H&E). The proliferation property of liver tissues was checked by immunohistochemistry staining with an antibody to PCNA (proliferating cell nuclear antigen). The HBsAg protein was stained with anti-HBsAg rabbit serum. Biotinylated anti-rabbit immunoglobulin G followed by avidin-biotin peroxidase was used for detection. Liver tissues from littermate mice were used as negative controls.
Total RNA was isolated using TRIzol reagent (Life Technologies, Carlsbad, CA) based on the suggested protocol. About 20 g of total RNA from each sample underwent electrophoresis on the 1% denaturing agarose gel and was transferred to a nitrocellulose membrane. The fixed membrane was then hybridized with the 32p-labeled probes to detect the specific gene expression.
For standard electron microscopic techniques, 1 mm3 of mouse liver was perfused with 3% glutaraldehyde in 0.075 M phosphate-buffered saline (PBS) (pH 7.4) at 4° C for 2 hours, then fixed with 1% osmic acid in 0.24 M PBS (pH 7.4) at 4° C for 2 hours. Sixty- to seventy-nanometer sections were stained with uranium acetate and plumbum citrate.
Generation of the HBsAg and HBx Gene Knock-in Transgenic Mice.
We generated two HBV gene knock-in transgenic mouse lines, each containing the complete genes of HBsAg and HBx. The targeting construct p21-HBsAg or p21-HBx (Fig. 1A) was used to introduce the HBsAg or HBx gene into the same p21 locus. Three ES clones containing a correct targeting event for each targeting construct were obtained out of 113 and 103 G418 and FIAU double resistant clones. Two targeted ES clones for each construct were injected into C57BL/6 blastocysts to obtain germline transmission. The offspring were tested for homologous recombination and the presence of the HBV gene insertions by Southern blotting (Fig. 1B) and PCR analysis (Fig. 1C). The transcript of p21 could be detected in wild type and both of the heterozygous transgenic mice (Fig. 1D). The mRNA of HBsAg could be detected in liver, kidney, and testis of the heterozygous transgenic mice, while the mRNA of HBx was only found in liver and kidney (Fig. 1E). The expression of HBsAg in liver started at E16 and continued for at least 24 months thereafter (data not shown). The mean serum glutamic-oxalacetic transaminase levels in mice at the ages of 5 months were 156.4 ± 10.8 IU/L in wild type control mice (n = 7), 458.7 ± 81.8 IU/L in p21-HBsAg transgenic mice (n = 7) (statistically significant, P = 0.01), 187.6 ± 23.3 IU/L in p21-HBx transgenic mice (n = 7) (statistically not significant). The mean serum glutamic-pyruvic transaminase levels in mice at the same ages were 62.3 ± 7.4 IU/L in wild type control mice, 122.3 ± 24.4 IU/L in p21-HBsAg transgenic mice (n = 7, statistically significant, P < 0.05), 84.9 ± 18.2 IU/L in p21-HBx transgenic mice (n = 7, statistically not significant), while serum total protein, albumin, triglyceride, cholesterol, and alkaline phosphatase levels were not changed significantly in both transgenic mice.
Development and Characteristics of HCC in HBV Gene Knock-in Transgenic Mice.
Transgenic mice were sacrificed 3, 6, 12, 15, 18, and 24 months after birth for pathologic analysis. We found both male p21HBsAg/+ heterozygotes (8/15, 53.3%) and p21HBsAg/HBsAg homozygotes (8/11, 72.7%) developed liver tumors between the ages of 15 and 24 months, while no female mice developed tumors at the same ages (Table 1). Most of the female (5/11, 45.5% in heterozygotes; 3/7, 42.9% in homozygotes) and male (6/10, 60.0% in heterozygotes; 7/11, 63.6% in homozygotes) p21-HBx transgenic mice developed liver tumors 18 months after birth or even much later. In contrast, none of the wild type littermate control mice of the same age developed HCC (see Table 1). The HCCs developed in transgenic mice are multifocal and are usually paler than the surrounding liver tissues. In most cases, one or two large tumors (0.5–2.5 cm) highly vascularized were dominant in the HBV transgenic livers, with many smaller tumors distributed throughout (Fig. 2A, B). Nearly half of mice (9/20) up to the age of 12 months had hepatic steatosis without inflammatory or neoplastic changes. However, male p21-HBsAg transgenic mice between the ages of 15 and 24 months showed that moderately well-differentiated HCCs made of eosinophilic cells compressed the neighboring nontumorous liver parenchyma (Fig. 2C). HCCs in male p21-HBsAg transgenic mice were usually marked with binucleated hepatocytes and PCNA-positive cells (Fig. 2E). Mitotic figures (Fig. 2G) and eosinophilic Mallory bodies (Fig. 2I) were also found in the transgenic liver nodules. Immunohistochemistry staining revealed HBsAg expressed in the cytoplasm of hepatocytes (Fig. 2K). HCCs compressing the neighboring normal hepatocytes (Fig. 2D, F) were developed in p21-HBx transgenic mice older than 18 months and were usually accompanied with hepatic steatosis and inflammation (Fig. 2H). Nuclear pleomorphism (Fig. 2J) was often found in hepatic nodules. Disorganized endoplasmic reticulum was also seen in HCCs of p21-HBx transgenic mice (Fig. 2L). There was no evidence of significant fibrosis within the affected livers. Metastases were not detected in either of the HBV gene knock-in transgenic mice.
Table 1. Incidence of Hepatic Tumors in HBV Gene Knock-in Transgenic Mice
Number of Mice
Mice With Hepatic Tumors
Mice were sacrificed and analyzed between the ages of 15 and 24 months. Data were statistically analyzed using independent samples t test with SPSS10.0 software (SPSS Inc., Chicago, IL).
Differential Expressed Genes in HCC Versus Liver of Transgenic Mice.
To study the molecular events during the development of HCC, we analyzed the gene expression profiles of liver tissues of transgenic and control mice with cDNA array analysis (data not shown). Some of the differential expressed genes at transcription levels were confirmed by Northern blot analysis. We found that Aldolase A, which is involved in the glycolytic pathway and increased in livers from patients with HCC,15 was up-regulated in liver tumors from both p21-HBsAg and p21-HBx transgenic mice. Meanwhile, metallothionein II was down-regulated (Fig. 3A). Previous studies have shown that increased mitotic phosphorylation of histone H3, a gene specifically expressed during the S phase of the cell cycle, is a major precipitating factor of chromosome instability.16 In this study, we found that histone 3 family 3a, one transcript of histone H3, was elevated in the liver tumors compared with the adjacently normal tissues, while p53 was down-regulated (Fig. 3A). The expression of the s3a ribosomal protein gene, which has been reported to be up-regulated in the tissues of human HCC,17 was also found to be slightly enhanced (Fig. 3A). These data indicate that the molecular changes in HBV gene knock-in transgenic mice largely resemble those of HCC in humans.
Up-regulated Expression of ER-β in Tumors From Male HBsAg Gene Knock-in Transgenic Mice.
Male gender is one of the risk factors of hepatic carcinogenesis in humans.1 Clinical studies have shown that mean serum estradiol levels are significantly higher among HCC patients compared with controls.18HBsAg-positive patients with variant estrogen receptor α (ER-α) were characterized by the worst survival.19 In this study, we checked the expression of androgen receptor, ER-α, and ER-β in HBV gene knock-in transgenic mice by Northern blot analysis. The expression of androgen receptor and ER-α had not been detected in livers of the HBV transgenic mice and controls (data not shown), but the expression of ER-β was extremely elevated at the transcriptional level only in the liver tumors developed in male p21-HBsAg transgenic mice (Fig. 3B). No transcripts of ER-β were detected in liver tumors developed in male or female p21-HBx transgenic mice or very aged (30 months old) female p21-HBsAg mice (Fig. 3B).
Analysis of the effects of HBV genes overexpressed in the liver of conventional transgenic mice showed that high expression of HBsAg and HBx in some background causes HCC,7, 8 while the HBsAg, core, and even the whole virus expression has no obvious pathologic effect in other lineages.9, 20, 21 Recent studies have indicated that chronic immune mediated liver cell injury triggers the development of HCC.22 However, the mechanism of the direct roles of long-term expression of HBsAg or HBx in the development of HCC is still debated. In these studies, we created two gene knock-in transgenic mouse strains harboring the HBsAg or HBx gene in the same locus to avoid possible integration site artifacts. We showed that both the HBsAg and HBx gene driven by their own promoter could express persistently during the lifetime of the HBV gene knock-in transgenic mice and eventually cause HCC. Previous studies have shown that p21-deficient mice develop spontaneous tumors at an average age of 16 months; however, none of 73 mice homozygously null for p21 over a 2-year period developed HCC, suggesting that p21 deficiency does not directly increase the susceptibility to HCC.23 The presence of HCC in heterozygous transgenic mice carrying a functional allele of p21 clearly demonstrated the function of HBsAg and HBx in hepatocarcinogenesis. On the other hand, the consequence of p21 deficiency in the HCC developed in the homozygous HBV gene knock-in transgenic mice still needs to be carefully clarified, because the loss of p21 enhances the ability of HBX to cause proliferation in hepatocytes.24
Many previous studies have shown that HBx could be a prime candidate for mediating HBV pathologic effects. HBx can act as a co-transactivator to induce cell apoptosis and inhibit the proliferation of liver cells during the development of HCC.25, 26 Our observations that HBsAg knock-in transgenic mice developed HCCs 3 months earlier than HBx transgenic mice demonstrated that HBsAg also plays an important direct role in the development of HBV-related HCC though a mechanism different than that of HBx. We noticed that only male HBsAg gene knock-in transgenic mice between the ages of 15 and 24 months developed HCCs. This observation is consistent with epidemiologic data showing that men chronically infected with HBV are more prone to liver cancer than women. This suggests that HBsAg is one of the HBV genes responsible for the higher numbers of males who get HCC. We found that ER-β was greatly up-regulated at the transcriptional level in the tumors from male p21-HBsAg transgenic mice. This indicated that the ER-β signaling pathway might be involved in the gender difference during the development of HBsAg-related HCCs. ER-β is a second estrogen receptor; the first was reported in 1996.27 Many studies have shown ER-β expressed in human prostate, breast,28 and gastric cancer29; however, the expression and significance of ER-β in hepatocarcinogenesis have not been reported. Our data provide evidence that ER-β may play important roles in the development of HBsAg related HCC. The finding that ER-β was only seen in fully malignant HBsAg tumors also suggested that ER-β oncogenic activity is limited to the late stages of HCC progression. It has been shown that ER-β could recruit steroid receptor coactivator 1 through phosphorylation by mitogen-activated phosphorylation kinase.30 It seemed likely that overexpression of ER-β could cause the changes in the levels or activity of cofactors and the expression of target genes that affect the genomic stability and differentiated status of hepatocytes and eventually contribute to the development of HCC. Results from antiestrogen therapy on inoperable HCC are conflicting.31, 32 This may be partially due to the low-level expression of ER-α in HCC. Our data suggest that ER-β–specific ligands may be useful targets for improving the treatment of HCC.
The authors thank Z. Li for providing plasmid pADR-1 carrying the genome of HBV and Yi Yang and Tao Zhou for electron microscopy.