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Carcinogenesis |
Banyu Tsukuba Research Institute (Merck), Tsukuba 300-2611 [N. H., H. M., H. O., N. M., M. O., Y. T.]; Laboratory of Biomedical Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033 [M. M. T.]; and Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501 [H. O., M. O., M. M. T.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Among them, HCC shows a high incidence of ß-catenin mutation at 20–30% (10
, 11)
, and mutations in the AXIN gene are also found occasionally (14)
, suggesting that the Wnt signaling pathway plays a critical role in hepatocarcinogenesis. Although the molecular mechanisms of hepatocarcinogenesis are not fully elucidated, inactivation of tumor suppressor genes with loss of heterozygosity has been reported for the p53 (15
, 16) , Rb1 (15)
, and APC gene loci (17)
. Elevated expressions of c-myc (18)
, cyclin D1 (19)
, transforming growth factor-
(20)
, and insulin-like growth factor II (21)
have also been found. Thus far, various transgenic mice have been established that overexpress activated oncogenes such as H-ras, c-myc (22)
, SV40 T-antigen (23)
, cyclin D1 (24)
, or core proteins of hepatitis C virus (25)
, resulting in hepatocellular carcinomas. On the other hand, there have been no mouse models of HCC that carry mutations in the Wnt signaling pathway genes in the liver.
We established previously a mouse strain containing a mutant ß-catenin allele, the exon 3 of which was sandwiched by loxP sequences [Catnblox(ex3) mouse] (26) . When Cre recombinase was expressed in the intestines, the GSK3ß phosphorylation sites were deleted, which caused adenomatous intestinal polyps. Recently, we found that Cre-mediated recombination occurred also in the liver in our Catnblox(ex3) mice crossed with cre-expressing transgenic mouse TgFabpl-cre. On the other hand, exon 3 deletion mutations have been reported in some carcinogen-induced HCC in the mouse (27 , 28) . To determine whether stabilized ß-catenin can cause HCC, we have introduced Cre into the liver of the Catnblox(ex3) mice by an adenovirus-mediated gene delivery system.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PCR Detection of ß-Catenin Exon 3 Deletion.
Cre-mediated deletion of ß-catenin gene exon 3 was confirmed by PCR, using primers BCAT-GF2 (5'-GGTAGGTGAAGCTCAGCGCAGAGC-3') and LXR1 (5'-GGCCAGTACTAGTGAACCTCTTCG-3'), for 35 cycles of 94°C for 30 s, 60°C for 1 min, and 72°C for 1 min. The wild-type gene was amplified as described previously (26)
.
Constructions of AdCMV-cre and AdCMV-lacZ.
The AdCMV-cre was constructed by inserting an expression cassette that contains the cre gene (29)
, controlled by the human CMV promoter, into a human type 5 and E1E3-deleted adenoviral vector. The bacterial lacZ gene was used to construct the AdCMV-lacZ (30)
. The recombinant adenovirus was amplified in 293 cells, purified by CsCl gradient centrifugation twice, and dialyzed. Infectious titers were determined using a TCID50 end point dilution method (31)
. A high titer of AdCMV-cre (1011 pfu/ml) was obtained. The diluted aliquots (100 µl) were injected into the tail vein of 7-week-old mice.
Western Immunoblot Analysis.
Tissue lysates were prepared by sonication in lysis buffer [10 mM HEPES (pH 7.8), 10 mM KCl, 0.1 mM EDTA, and 0.1% NP40] containing protease inhibitors (phenylmethylsulfonyl fluoride, aprotinin, pepstatin, leupeptin, and antipain.) Aliquots of 50 µg of total protein were resolved by SDS-PAGE, transferred to a membrane, and detected by a rabbit polyclonal anti-ß-catenin antibody (Sigma Chemical Co., St. Louis, MO) coupled with the ECL detection system (Amersham-Pharmacia Biotech, Inc., Piscataway, NJ).
Histology and Immunohistochemical Analyses.
Sections were prepared and stained with H&E as described previously (26)
. For immunohistochemistry, a rabbit polyclonal anti-ß-catenin antibody (Sigma Chemical Co.) was used at a dilution of 1:500. The primary antibody was detected using the Elite ABC rabbit kit (Vector Laboratories, Burlingame, CA) as described (26)
.
Electron Microscopy.
Samples were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C for 2 h, washed in phosphate buffer (pH 7.2), treated with 1% osmium in 0.1 M phosphate buffer (pH 7.2) at 4°C for 2 h, and dehydrated in graded ethanols, followed by embedding in an epoxy resin. The sample sections (70 nm) were prepared, stained by uranylacetate, and examined under an electron microscope at BML (Kawagoé, Saitama, Japan).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ex3; Ref. 26
). The mutation was generated by Cre-mediated excision of exon 3 of the ß-catenin gene (Catnb) that encodes codons 5–81, including the serine and threonine residues phosphorylated by GSK3ß. To introduce Cre into the intestinal epithelium, we constructed two types of cre-expressing mice, cytokeratin 19-cre knock-in mice and fatty acid binding protein (Fabpl)-cre transgenic mice, and crossed them with the Catnblox(ex3) mice, respectively. Numerous intestinal polyps formed in both kinds of compound heterozygous mice (26)
. Interestingly, however, the body weight of the [Catnblox(ex3):TgFabpl-cre] mice started to decrease at the third week after birth, and the mice died within 4–5 weeks, whereas the littermate wild-type and single heterozygous mice were normal and healthy (Fig. 1A)
. It is unlikely that the early death of the TgFabpl-cre compound heterozygotes was attributable to the intestinal polyposis, because the cytokeratin 19-cre compound heterozygotes survived >3 months, despite their severer polyposis (26)
.
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. Upon Western immunoblot analysis, the mutant ß-catenin protein was detected only in the small intestine and liver (Fig. 1C)
but not in other organs (data not shown). It is interesting that the band for the mutant ß-catenin (ß-cateninex3) was stronger in the liver than in the small intestine, suggesting that recombination took place in a substantial fraction of the hepatocytes.
Upon histological analysis, numerous vacuoles were found in the hepatocytes of 4-week-old compound heterozygotes [Catnblox(ex3):TgFabpl-cre] but not in those of the age-matched single-heterozygous or wild-type mice (Fig. 1D)
. Vacuoles of various sizes were prominent, especially in the periportal areas. It is worth noting that such degenerative changes were not observed before 3 weeks of age, although mutant ß-catenin was already detected in the liver at the first week after birth (data not shown; see "Discussion"). It has been reported that fatty changes of the hepatocytes are often observed in both atypical adenomatous hyperplasia and well-differentiated hepatocellular carcinomas (32, 33, 34)
. To determine whether the vacuoles contained lipids, frozen sections were stained with Oil Red-O, Sudan Black B, and Nile Blue, respectively. However, no staining was detected with either dye, ruling out lipid accumulations in the vacuoles. An electron microscopic analysis revealed that these vacuoles were swollen mitochondria (data not shown), similar to the Catnblox(ex3) mice infected with the Cre-expressing adenovirus (see below).
Hepatomegaly in the Cre-expressing Adenovirus-infected ß-Catenin Mutant Mice, Catnblox(ex3).
Recombinant adenovirus vectors have been successfully used to express Cre in transgenic mice (35
, 36)
. To investigate the possible role of the stabilized ß-catenin in hepatocarcinogenesis more directly, we constructed recombinant adenovirus strains for expressions of the cre gene (AdCMV-cre; Fig. 2A
) and the bacterial ß-galactosidase gene (AdCMV-lacZ), respectively, both driven by the human CMV promoter. When AdCMV-lacZ was inoculated into mice through the tail vein, LacZ expression was detected only in the liver but not in other organs (data not shown). The result is consistent with earlier reports (35
, 36)
.
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. Although both mouse strains appeared normal and healthy immediately after the inoculations, the Catnblox(ex3) mice started to die at day 12 after infection, and more than half of them died by day 16, whereas the control C57BL/6N mice remained healthy throughout (Fig. 2C)
. When three littermate wild-type mice were infected as additional controls, they also remained normal and healthy for about 1 year (data not shown).
At day 18 after infection, the surviving Catnblox(ex3) and control mice were necropsied. Markedly enlarged livers were found (i.e., hepatomegaly) in the Catnblox(ex3) mice, although no other gross abnormalities were detected in either Catnblox(ex3) or control mice (Fig. 2D)
. The weight of the infected Catnblox(ex3) liver was 3.23 ± 0.42 g, which was more than twice heavier than that of the control mice, 1.35 ± 0.06 g (Fig. 2E
; n = 4; P < 0.001). No hepatomegaly or decrease in the survival rate was observed when Catnblox(ex3) mice were infected with 109 pfu/mouse AdCMV-lacZ virus (data not shown), which ruled out the possibility that the hepatomegaly was caused by the adenovirus infection to the Catnblox(ex3) mice. Because hepatocellular damage is often associated clinically with increased serum enzyme levels (37)
, we performed a biochemical analysis of the serum samples taken upon necropsy. The serum alanine aminotransferase level was increased by 2–3 times in the Catnblox(ex3) mice infected with AdCMV-cre compared with those from the infected wild-type mice. Other enzymes such as aspartate aminotransferase and leucine aminopeptidase levels were also increased by two to three times. At the same time, the levels of certain globulin families were decreased or increased by 20–40% in the serum of the Catnblox(ex3) mice, reflecting the acute hepatocyte injury (data not shown). To confirm the Cre-mediated recombination in the liver, the genomic DNA and cell lysate were prepared and subjected to PCR and Western immunoblot analyses, respectively. As shown in Fig. 2, F and G
, respectively, both the recombination in the ß-catenin gene and the expression of the mutant ß-catenin protein were detected at both days 5 and 18 after infection. These results indicated that the hepatomegaly was caused by the mutant and therefore stabilized ß-catenin.
Mitochondrial Swelling in the Cre-expressing, Adenovirus-infected ß-Catenin Mutant Mice.
To determine whether the hepatomegaly was caused by neoplastic changes, we then analyzed the liver histologically. As shown in Fig. 3, A and B
, many hepatocytes of the infected Catnblox(ex3) mice showed ballooning changes, although the lobular architecture consisting of the central vein, portal vein, and bile duct remained unaffected. Although the cell multiplication rate monitored by the bromodeoxyuridine uptake and proliferating cell nuclear antigen labeling index were increased in the liver by adenovirus infection itself (determined at day 5 and/or day 18 after infection), there was no significant difference in these parameters between the Catnblox(ex3) and the wild-type controls (data not shown; see "Discussion"). Interestingly, numerous vacuoles that were similar to those found in the [Catnblox(ex3): TgFabpl-cre] mice were found in the AdCMV-cre-infected Catnblox(ex3) mice but not in the infected wild-type controls (Fig. 3, A and B)
. Upon an ultrastructural examination with an electron microscope, crista structures were observed in the vacuoles, which strongly suggested that the vacuoles were swollen mitochondria (Fig. 3, C and D)
. It should be noted that mitochondrial swelling was not detected in the adjacent normal hepatocytes that could have remained uninfected with AdCMV-cre (Fig. 3C)
. To rule out the possibility that swollen mitochondria were the result of apoptotic changes, we performed terminal deoxynucleotidyl transferase-mediated nick end labeling assays for DNA fragmentation as well as an immunodetection for the activated caspase 3, but the both results were negative (data not shown). These observations collectively indicate that acute hepatocyte damages occurred in the infected liver.
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(41)
, and that nuclear localization of ß-catenin is found in some hepatocellular carcinomas (42)
. Accordingly, we next determined the localization of ß-catenin in the AdCMV-cre-infected liver by immunohistochemical staining with an anti-ß-catenin antibody. At day 5 after infection, the cytoplasmic and nuclear immunoreactivities were elevated in the Catnblox(ex3) mice compared with those in the control mice (Fig. 3, E and F)
. This result is consistent with that of the Western immunoblotting analysis (Fig. 2G)
. At day 16 after infection, however, no nuclear localization was detected in the hepatocytes with mitochondrial swelling, but staining was found only at the membrane (data not shown). These data suggested that nuclear localization of stabilized ß-catenin was detected at day 5 after infection but lost by day 16, probably because of CTL-mediated immune responses, in this high multiplicity infection.
No HCC in the ß-Catenin Mutant Mice Infected with AdCMV-Cre.
Mutant ß-catenin was expressed in nearly half of the hepatocytes by infection with 109 pfu/mouse AdCMV-cre and in almost all hepatocytes by crossing with the TgFabpl-cre transgenic mice. Such a high frequency mutation does not necessarily reflect the clinical conditions that lead to hepatocellular carcinogenesis. In addition, Catnblox(ex3) mice infected with 109 pfu/mouse AdCMV-cre died within 3 weeks, which prevented us from following up their hepatic tumorigenicity for longer periods.
To overcome these problems and to mimic more clinical situations, lower titers of AdCMV-cre at 108 and 107 pfu/mouse, respectively, were inoculated to the Catnblox(ex3) mice and compared with the high multiplicity infection results. One of the infected mice for each virus multiplicity was necropsied at day 3 after infection, and genomic DNA and cell lysate were analyzed. As shown in Fig. 4A
, the PCR band for the recombined ß-catenin allele was detected in dose-dependent intensities. Upon Western immunoblot analysis, however, the mutant ß-catenin protein could be detected only in the 109 pfu-infected livers, possibly because of the lower sensitivity of the antibody than that of the PCR (Fig. 4B)
. As expected, the mice infected with 107 or 108 pfu AdCMV-cre survived >6 months and remained normal and healthy. Upon necropsy, no hepatomegaly was found. However, the Cre-recombined allele was detected in the 108 pfu-infected mouse livers by PCR, even at 6 months after infection (Fig. 4C)
, suggesting that the virus-infected hepatocytes were not completely eliminated, but a few cells were still surviving in the liver. Nevertheless, neither hyperplastic changes nor dysplastic foci were detected histologically, although >30 Catnblox(ex3) mice were examined (data not shown). Interestingly, upon immunohistochemical analysis, nuclear localization of ß-catenin was detected in some hepatocytes only in the Catnblox(ex3) mice (Fig. 4E)
but not in the wild-type controls (Fig. 4D)
. These hepatocytes appeared healthy and quiescent, where no vacuoles were detected. Taken together, these results strongly suggest that nuclear accumulation of ß-catenin alone is not sufficient for hepatocellular carcinogenesis, but additional mutations or epigenetic changes are required.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To rule out the possibility that swollen mitochondria were the result of apoptotic changes, we performed terminal deoxynucleotidyl transferase-mediated nick end labeling assays for DNA fragmentation as well as immunodetection for activated caspase 3, but both results were negative (data not shown). Mitochondrial swelling is often observed in acute virus hepatitis (43) , alcohol injury (44) , and treatment with peroxisome proliferators (45) , which have been identified as major risk factors for hepatocellular carcinogenesis. In the Catnblox(ex3) mice infected with AdCMV-cre at lower multiplicity, we could investigate both the acute and chronic phases of the viral infection, because the mice survived >6 months. At day 5 after infection, some mitotic figures were observed in both the Catnblox(ex3) and control mice. The bromodeoxyuridine uptake and proliferating cell nuclear antigen labeling index were also increased. Although it has been reported that a CTL-mediated immune response eliminates adenovirus-infected hepatocytes by day 21 after infection (46) , certain populations of the infected hepatocytes remained even after 6 months, because the recombinant mutant ß-catenin allele was detected by PCR. In addition, strong nuclear localization of ß-catenin was detected in some morphologically normal hepatocytes, which was not observed in the control mice. These results suggest that acute inflammatory reactions, subsequent clearance, and regeneration of the infected hepatocytes took place, but some infected cells survived through these changes.
There are some possible explanations why Catnblox(ex3) mice did not form any HCC foci:
(a) For hepatocellular carcinogenesis, a ß-catenin gene mutation may be insufficient, and additional changes may be required. In contrast, inactivation of Apc or stabilization of ß-catenin is sufficient to cause intestinal polyposis (26 , 47 , 48) . Nevertheless, expression of COX-2 in the stromal cells is required for further expansion of the intestinal polyps (49) . It is conceivable that additional mutations are necessary for hepatocytes to form neoplastic foci because the stromal microenvironment is different for the hepatocytes from the intestinal epithelium.
(b) It is possible that stabilization of ß-catenin may not be necessary in the early stage of hepatocellular carcinogenesis but important in a later stage. In the diethylnitrosamine-induced mouse HCC model, mutations in the ß-catenin gene were found in HCC but not in adenomas (42) . Although ß-catenin mutations were reported as an early event in some chemically induced mouse HCCs (27) , it is conceivable that exon 3 deletions play a different role from missense mutations, such as progression into hepatoblastomas (28) .
Recently, a transgenic mouse line was reported in which an NH2-terminal-truncated, dominant mutant N131 ß-catenin was expressed under the control of a liver-specific enhancer of the aldorase B gene (50)
. The mice showed marked hepatomegaly soon after birth, similar to that induced by AdCMV-cre at high multiplicity in our model, but they also failed to form any neoplastic foci in the liver. Although the phenotypes of this transgenic strain are somewhat similar to those of our virus infection model, there are distinct differences as well. In their transgenic liver, no vacuolar degeneration or mitochondrial swelling in the hepatocytes was observed. On the other hand, foci of hepatocellular hyperplasia were reported that were not found in the AdCMV-cre-infected mice. These differences may be explained by several reasons. Although the N131 ß-catenin lacks the -catenin binding domain, ß-cateninex3 retains it (51)
. Whereas the N131 transgene is expressed throughout the embryonic and postnatal development, the AdCMV-cre virus is inoculated after weaning and causes acute inductions of ß-cateninex3.
In conclusion, we have established a mouse strain in which a dominant and stable ß-catenin mutation was conditionally introduced into the liver. Although these mice showed a transient hepatocyte hyperplasia, they did not develop any neoplastic foci. These results suggest that stabilization of ß-catenin is not sufficient for hepatocarcinogenesis, and that additional mutations or epigenetic changes may be required.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by the Joint Research Fund between the University of Tokyo and Banyu Pharmaceutical Co. and grants from Monbusho (MESSC) and the Organization for Pharmaceutical Safety and Research (OPSR), Japan (to M. M. T.). 
2 To whom requests for reprints should be addressed, at Department of Pharmacology, Graduate School of Medicine, Kyoto University, Yoshida-Konoé-cho, Sakyo, Kyoto 606-8501, Japan. Phone: 81-75-753-4402; Fax: 81-75-753-4391; E-mail: taketo{at}mfour.med.kyoto-u.ac.jp
3 The abbreviations used are: GSK3ß, glycogen synthase kinase 3ß; HCC, hepatocellular carcinoma; CMV, cytomegalovirus; pfu, plaque-forming unit(s).
Received 9/14/01. Accepted 2/14/02.
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REFERENCES |
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, mean body weights of the Catnblox(ex3):TgFabpl-cre mice (lox/cre) with SD (bars; n = 4). 
, body weights of the simple heterozygotes [i.e., Catnblox(ex3) and TgFabpl-cre, respectively] and the wild-type mice combined (control), with SD (bars; n = 7). B, PCR analysis of various organs for the Cre-mediated deletion of the ß-catenin gene exon 3. Each lane was loaded with the PCR product amplified on the DNA from the brain (Br), thymus (Thy), heart (Hrt), lung (Lu), stomach (St), small intestine (Int), colon (Co), liver (Li), spleen (Sp), kidney (Ki), testis (Tes), and pancreas (Pa). Right, position for the deleted ß-catenin allele (
, packaging sequence; CMV, human CMV early gene promoter; pA, polyadenylation sequence. See Chartier et al. 
