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Overexpression of Glutamine Synthetase Is Associated with ß-Catenin-Mutations in Mouse Liver Tumors during Promotion of Hepatocarcinogenesis by Phenobarbital¹

Institut für Toxikologie, Universität Tübingen, 72074 Tübingen [S. L., D. S., A. B., M. S.]; Institut für Biochemie, Universität Leipzig, 04103 Leipzig [F. G., R. G.]; and Universitäts-Frauenklinik, Universität Tübingen, 72076 Tübingen [R. K.], Germany

	ABSTRACT

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Phenobarbital (PB) is an antiepileptic drug that promotes hepatocarcinogenesis in rodents when administered subsequent to an initiating carcinogen like N-nitrosodiethylamine (DEN). In the mouse, the promotional effect of PB on liver tumor development results from a selective stimulation of clonal outgrowth of hepatocytes harboring activating mutations in the ß-catenin gene. Because glutamine synthetase (GS) has recently been shown to be a putative transcriptional target of ß-catenin, expression of GS during PB-mediated promotion of mouse hepatocarcinogenesis was investigated. Preneoplastic and neoplastic liver lesions were induced in 6-week-old male mice by a single injection of 90 µg/g body weight of DEN, and groups of mice were subsequently kept on PB-containing (0.05%) or control diet for 39 weeks. In PB-treated mice, 46 of 51 lesions (90%) were GS-positive in contrast to only 16 of 46 (35%) in mice not treated with PB. Approximately 33% of liver was occupied by neoplastic tissue in PB-treated mice, of which >80% was GS positive. By contrast, only 3.5% of liver consisted of neoplastic tissue in mice treated with DEN only, and 25% of this was GS positive. We have previously shown that ß-catenin mutations are present in 80% of liver tumors from PB-treated mice but are absent in liver tumors from mice treated with DEN only. By analyzing a panel of larger liver tumors, we now observed that tumors harboring ß-catenin mutations were GS positive, whereas tumors without ß-catenin mutations were GS negative. Similarly, tumors from an additional mouse carcinogenicity experiment where PB inhibited rather than promoted hepatocarcinogenesis were mostly GS negative. These data suggest that promotion of hepatocarcinogenesis by PB confers ß-catenin-mutated tumor cells with a selective advantage by up-regulation of GS expression.

	INTRODUCTION

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The enzyme GS³ (E.C.6.3.1.2) plays a key role in ammonia metabolism. It is highly expressed in liver, but only hepatocytes in the pericentral zone surrounding the central veins of the liver lobuli express the enzyme (1) . Most likely pretranslational mechanisms contribute to the regulation of zonation of GS; cis-regulatory elements within the GS gene involved in hepatocyte-specific expression of the enzyme have been identified (2 , 3) . Although heterogeneous expression of GS within the liver lobule is generally very stable and not changed under different physiological conditions, GS mRNA and protein were found to be elevated in human primary liver cancers (4, 5, 6) , and GS has been reported to be overexpressed in a small subpopulation of preneoplastic foci, adenomas, and carcinomas induced in rat liver by DEN or 2-acetylaminofluorene (7) .

The mechanisms of regulation of GS expression in hepatocytes are only partly understood; very recently, protein phosphatase 2A and ß-catenin were found to be involved in the regulation of hepatocellular GS (8) , and GS has been identified as one of the target genes strongly induced in livers of mice overexpressing a truncated oncogenic form of ß-catenin (9) .⁴ The ß-catenin gene codes for a protein associated with the intracellular domains of E-cadherin, a component of the adherens junction (for a recent review see Ref. 10 ). ß-Catenin is also essential in the Wnt/Wingless signaling pathway and mediates nuclear responses to Wnt signals by interacting with TCF (also known as lymphocyte enhancer-binding factor) transcription factors (10) . ß-Catenin levels are negatively regulated by phosphorylation of particular serine/threonine residues at the NH₂-terminal region mediated by glycogen synthase kinase 3ß. Phosphorylation by glycogen synthase kinase 3ß is catalyzed by a molecular complex, including among others, axin1 and the adenomatous polyposis coli protein and targets ß-catenin for ubiquitin/proteasome-mediated degradation. Inactivation of adenomatous polyposis coli or mutation of ß-catenin may lead to increased cytoplasmic concentration of ß-catenin, turning the protein into a potentially transforming form (10) .

We have recently shown that 80% of tumors generated in livers of mice treated at adult age with DEN, followed by continuous administration of the liver tumor promoter PB in their diet, harbor activating mutation⁵ in ß-catenin, whereas no such mutations were detected in tumors from mice treated with DEN alone (11) . This demonstrated that the nonmutagenic tumor promoter PB selects for ß-catenin-mutated hepatocytes during the promotional phase of hepatocarcinogenesis, potentially through modulation of Wnt/ß-catenin/TCF signaling. Therefore, we hypothesized that tumor promotion by PB in mouse liver may involve transcriptional activation of GS by activated ß-catenin, a potential mechanism that was investigated in the present study.

	MATERIALS AND METHODS

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Tissue Samples.
Mouse liver samples were obtained from two previous experiments with Cx32 wild-type and knockout mice (C57BL/129Sv x C3H/He) that were aimed to investigate the role of Cx32 in tumor promotion by PB (12) . Only samples from animals with Cx32 wild-type genotype were used in this study. In experiment 1, mice were given i.p. injections with a single dose of DEN (90 µg/g of body wt.) at 6 weeks of age, whereas 10 µg/g body weight of the carcinogen were injected into 12–15-day-old mice in the second experiment. DEN-treated mice were then kept on standard diet or on diet containing 0.05% PB until sacrifice, which was 39 weeks (experiment 1) or 25 weeks (experiment 2) after start of PB treatment, respectively. Macroscopically visible liver tumors (diameters > 3 mm) were collected and frozen in liquid nitrogen; the remaining livers were frozen on blocks of dry ice and stored at -80°C.

Mutation Analysis.
For screening for ß-catenin-mutations in mouse liver lesions, DNA was isolated from larger tumors or tissue samples were taken from glucose-6-phosphatase-stained liver sections by use of punching cannuli (13) and used for PCR amplification and direct sequencing of a 1473-bp fragment (isolated DNA) or a 297-bp fragment (tissue samples), both containing exon 2 of the ß-catenin gene as described previously (11) . Each mutation detected was confirmed by at least one independent PCR/mutation analysis.

Immunohistochemistry, Stereology, and Western Analysis.
GS was stained immunohistochemically in frozen liver sections (10 µm) by standard protocols (12) using anti-GS-rabbit antiserum (1:2000; Ref. 7 ), antirabbit IgG secondary antibody (Sigma-Aldrich, Taufkirchen, Germany), and 3-amino-9-ethylcarbazole/H₂O₂ as a substrate. Tumor cell proliferation was determined by use of a monoclonal rat anti-Ki-67 antibody (TEC-3; Dianova, Hamburg, Germany), which detects a nuclear antigen that is expressed at all phases of the cell cycle, except in G₀. Binding of antibody (1:20 dilution) to frozen liver sections was visualized using the appropriate horseradish peroxidase-conjugated secondary antibody as described above.

The area of tissue sections and of GS-positive and GS-negative lesions therein was quantitated by a computer-assisted digitizer system (14) .

Western blots with antibodies against GS (1:2000 dilution) and ß-catenin (catalog no. C19220; Transduction laboratories, Lexington, KY; 1:500) were performed as described previously (11) . Antibody binding was visualized using appropriate alkaline-phosphatase-conjugated secondary antibodies and CDP-Star as a substrate (Tropix; PE Applied Biosystems, Weiterstadt, Germany). Chemiluminescence signals were monitored by use of a CCD video camera system and quantified by use of TINA V2.09a software (Raytest, Straubenhardt, Germany).

Northern Analysis.
GS mRNA levels were determined by Northern analysis. RNA was isolated by Trizol Reagent (Life Technologies, Inc., Eggenstein, Germany) according to the manufacturer’s protocol, separated on a 1% denaturing agarose gel, and transferred to a nitrocellulose membrane by standard procedures. The blot was hybridized with a ³²P-labeled GS-specific cDNA probe.

	RESULTS

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The liver tumors used in this study were from two previously performed experiments aimed to investigate the mechanism of tumor promotion by PB (12) . In experiment 1, mice were given a single injection of DEN at the age of 6 weeks, and groups of mice were subsequently kept on PB-containing or control diet until sacrifice. Under these conditions, PB significantly promoted hepatocarcinogenesis (12) . In contrast, in experiment 2, where infant instead of adult mice were treated with DEN followed by treatment with PB-containing or control diet, an inhibitory rather than a promotional effect of PB on hepatocarcinogenesis was obtained (12) . The mechanism of this paradoxical effect of the barbiturate is not entirely clear, but similar effects have been repeatedly reported (Ref. 15 and references therein).

The expression of GS was first studied at the protein level by immunohistochemistry using frozen liver sections from PB-treated and -untreated mice of experiment 1 (promotion study). In the normal liver tissue, GS staining was limited to a small percentage of hepatocytes surrounding the terminal central veins, a staining pattern that has been repeatedly described before (1) . Often, the ring of GS-positive hepatocytes surrounding the central veins was interrupted by GS-negative cells (Fig. 1A) , which is also a characteristic feature of DEN-induced hepatocarcinogenesis in the rat (7) . Although most tumors from mice of experiment 1 not treated with PB (DEN only) showed no positive GS immunoreaction, the majority of neoplastic lesions in livers from their PB-treated counterparts were GS-positive: most tumors showed a more or less homogeneous GS-staining pattern, although heterogeneous expression of GS was also observed in some of the tumors (Fig. 1) . Data resulting from a quantitative analysis of GS-positive and GS-negative neoplastic lesions observed in experiment 1 are presented in Table 1 . In PB-treated mice (n = 7), 46 of 51 lesions (90%) were GS positive in contrast to only 16 of 46 (35%) in mice not treated with PB (n = 16). About 33% of liver was occupied by neoplastic tissue in PB-treated mice, of which >80% was GS positive. By contrast, only 3.5% of liver consisted of neoplastic tissue in mice treated with DEN only, and 25% of this was GS positive. These results indicate that PB-mediated promotion of hepatocarcinogenesis in mice is associated with a selection for GS-positive tumor populations.

View larger version (169K): [in this window] [in a new window]   Fig. 1. Expression of GS protein in neoplastic mouse liver lesions. A, the top panel shows an overview of a liver section from a DEN/PB-treated mouse from experiment 1 (promotion study) stained immunohistochemically for GS. Of 12 focal lesions in this section, 11 were GS positive; one GS-negative tumor transection is marked by white arrows. The bottom panel of the figure shows one of the GS-positive lesions at a higher magnification. Within the normal liver tissue, a terminal hepatic venule surrounded by GS-positive hepatocytes is visible; note the disruption of the GS-positive ring of cells around the venule, indicated by the arrow. Bar: 0.1 mm. B, the top panel shows the overview of a liver section from a DEN/PB-treated mouse from experiment 2 (inhibition study). Arrows indicate the outlines of three GS-negative liver tumors; note the regular pattern of GS-positive pericentrally located hepatocytes within the normal liver tissue and the almost complete absence of GS-positive cells within the tumor areas. The bottom panel of the figure shows an indicated area of one of the tumors at a higher magnification (H&E stain). Bar: 0.2 mm.

By screening of liver tumors from experiment 1 for mutations in the ß-catenin gene, we have recently observed that 80% of neoplastic lesions from DEN/PB-treated mice harbor base substitutions in the ß-catenin gene, whereas no such mutations were detected in tumors from mice treated with DEN alone (11) . Because GS was very recently found to be transcriptionally activated in hepatocytes expressing a transgenic-activated (truncated) form of ß-catenin in vitro and in vivo (9) , we hypothesized that there might be a link between mutation of ß-catenin and increased expression of GS during PB-mediated promotion of hepatocarcinogenesis in mice. Therefore, we used Western analysis to quantify the levels of GS in ß-catenin-mutated (n = 6) and ß-catenin wild-type (n = 6) tumors and normal liver isolated from mice that had been treated with and without PB during the promotional phase of hepatocarcinogenesis (for representative examples, see Fig. 2A ). In the normal liver tissues, there were no significant differences in GS protein levels between PB-treated and -untreated mice. By contrast, ß-catenin-mutated liver tumors from PB-treated mice showed >20-fold increases in GS protein as compared with ß-catenin wild-type tumors. Interestingly, GS-specific signals in liver tumors from mice without detectable ß-catenin mutations were almost undetectable and much lower than in normal liver, which is likely attributable to the almost complete lack of GS-positive normal hepatocytes in the tumorous areas (see also Fig. 1B ). These results strongly pointed toward a causal link between mutational activation of ß-catenin and enhanced expression of GS. In accordance with what we observed earlier (11) , the overall concentration of ß-catenin protein was not significantly enhanced in ß-catenin-mutated tumors as compared with wild-type tumors (Fig. 2A) . This observation suggests that the protein expressed by the mutated form of ß-catenin, but not wild-type ß-catenin, has the potential to activate GS expression, either directly or indirectly.

View larger version (70K): [in this window] [in a new window]   Fig. 2. Expression of GS and ß-catenin in ß-catenin-mutated and wild-type mouse liver tumors. A, Western blot analysis of GS and ß-catenin expression. Antibody binding was visualized using alkaline phosphatase-conjugated secondary antibodies and CDP-Star as a substrate; chemiluminescence signals were monitored by use of a video camera system. Examples shown are representative for a total of six tumors and three normal tissues from each treatment group. B, Northern blot analysis of GS mRNA. Total RNA was isolated from liver tumors characterized for the presence or absence of ß-catenin mutations and from normal liver (N.L.) of PB-treated and -untreated mice and subjected to Northern analysis using a 32P-labeled GS-specific cDNA probe. The loading control shows 18S and 28S RNA banding in the ethidium bromide-stained gel.

Transcriptional activation of GS may contribute to increased proliferation of hepatoma cells (7) . Therefore, we investigated by immunohistochemistry the Ki-67-labeling index of GS-positive and GS-negative tumors in livers from PB-treated mice of experiment 1. One example is given in Fig. 3 , demonstrating two tumors with opposing GS phenotype within one liver. While no ß-catenin mutation was detectable in the GS-negative tumor (a in Fig. 3A ), the GS-positive tumor (b in Fig. 3A ) demonstrated a T:AG:C transversion in codon 35 of ß-catenin (Fig. 3C) , a type of mutation that has been detected in human hepatocellular cancer.⁵ The ß-catenin-mutated and GS-positive tumor showed a higher Ki-67-labeling index as compared with the GS-negative tumor (Fig. 3B) . The mean Ki-67-labeling index (irrespective of PB treatment of mice) of GS-positive tumors analyzed was 3.62 ± 1.55% (n = 23), whereas GS-negative lesions showed a mean labeling index of 2.89 ± 1.74% (n = 12). Because of large variation in labeling indices, this difference, however, was not statistically significant (P = 0.068; Mann-Whitney test).

View larger version (74K): [in this window] [in a new window]   Fig. 3. Differences in GS staining and Ki-67-labeling index in two tumors with and without ß-catenin mutations. A, overview of a liver section from a DEN/PB-treated mouse from experiment 1 stained immunohistochemically for GS; note that tumor a lacks positive GS staining, whereas tumor b shows a positive GS immunoreaction. B, left panel: tumor b at higher magnification (bar: 0.02 mm) immunostained for Ki-67; open arrows point toward Ki-67-positive nuclei; right panel: Ki-67-labeling index of tumors a and b. C, sequence analysis of ß-catenin; multiple samples were taken from tumors a and b by punching cannuli and directly used for PCR-based amplification of ß-catenin exon 2 followed by direct sequencing. The arrow indicates a single base substitution in ß-catenin codon 35 in tumor b.

	DISCUSSION

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GS mRNA levels have recently been reported to be elevated in livers of transgenic mice harboring an activated ß-catenin transgene, suggesting that ß-catenin signaling may be involved in the control of GS expression (9) . Similarly, the results of this study establish a clear association between GS expression and mutation of the ß-catenin gene in mouse liver tumors generated by an initiation/promotion regimen, including PB and increased expression of GS was found to result from transcriptional activation of the gene within tumor cells containing activated ß-catenin. Mutation or truncation of ß-catenin may lead to increased cytoplasmic levels, nuclear translocation, and activation as a transcription factor (10) . However, there was no indication from previous (11) or present work for such an increase in intracellular ß-catenin concentration or nuclear accumulation of the protein in ß-catenin-mutated liver lesions. Moreover, cyclin D1 and c-myc, both well-known target genes transcriptionally activated by ß-catenin/TCF (10) , did not differ in expression between ß-catenin-mutated and wild-type tumors (11) . Similarly, neither of the two genes was increased in expression in livers of ß-catenin transgenic mice (19) . This indicates that mutated ß-catenin signaling in mouse liver selectively targets GS gene-regulatory elements by other mechanisms currently investigated in our laboratories.

PB has been shown to enhance hepatocarcinogenesis in mice initiated by DEN at adult age but inhibited tumor formation when given to mice initiated at infancy (Ref. 15 and references therein). Similar results were observed in the two experiments from which material was used in this investigation (12) . This paradoxical action of PB, acting as promoter or inhibitor of carcinogenesis, depending on age of mice at initiation, enabled us to distinguish effects of the barbiturate specific for tumor promotion from those more generally evoked by the drug. The results of this study very clearly show that selection for GS-positive neoplastic lesions in mouse liver occurs almost exclusively under the experimental conditions where PB promoted hepatocarcinogenesis, whereas <2% of neoplastic lesions produced under the PB-suppressive conditions were GS positive. This result indicates that selection for GS-positive clones is a hallmark of tumor promotion by the barbiturate.

Recently, it has been shown that enhanced expression of PB-inducible target genes such as cytochrome P450 enzymes involves activation of the nuclear receptor CAR that binds together with retinoic acid X receptor as heterodimeric transcription factor CAR response elements in PB-responsive genes. Whether CAR is also required for promotional effects of PB is not known, but experiments with CAR knockout mice (20) might potentially answer this question.

	ACKNOWLEDGMENTS

 
We thank Johanna Mahr, Elke Zabinsky, and Verene Henkel for their excellent technical assistance. We also thank Dr. Christoph Köhle for helpful discussions.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported, in part, by the Deutsche Forschungsgemeinschaft Grant SCHW 329/3-1 and the Tübinger Fortüne Program Grant 908-0-0.

² To whom requests for reprints should be addressed, at Institute of Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany. Phone: 49-7071-29-77398; Fax: 49-7071-29-2273; E-mail: michael.schwarz{at}uni-tuebingen.de

³ The abbreviations used are: GS, glutamine synthetase; DEN, N-nitrosodiethylamine; TCF, T-cell factor; PB, phenobarbital; Cx32, connexin32; CAR, constitutive androstane receptor.

⁴ F. Gaunitz and R. Gebhardt, unpublished observation.

⁵ Internet address: www.stanford.edu/rnusse/wntwindow.html.

Received 2/15/02. Accepted 8/ 8/02.

	REFERENCES

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