Predominant mutation of codon 41 of the ß-catenin proto-oncogene in rat colon tumors induced by 1,2-dimethylhydrazine using a complete carcinogenic protocol
Robert Koesters,2,
Margrit A. Hans,
Axel Benner1,
Rüdiger Prosst,
Johannes Boehm,
Johannes Gahlen and
Magnus von Knebel Doeberitz
Department of Surgery, University Hospital of Heidelberg, INF 110, 69120, Heidelberg and
1 Central Unit of Biostatistics, Deutsches Krebsforschungszentrum, Heidelberg, Germany
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Abstract
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Constitutive activation of the wnt-signaling pathway plays an important role during both human and rat colon carcinogenesis and can be brought through mutations in either the adenomatous polyposis coli or the ß-catenin gene. Mutations found in the ß-catenin gene typically affect one out of four regulatory phosphorylation sites near the N-terminus of the ß-catenin protein. Whereas in human colon cancers, however, the majority of ß-catenin mutations directly alter threonine 41 or serine 45; the ß-catenin mutations found in chemically induced rat colon tumors seemed to cluster around codon 33 instead. Unlike previous studies, that have used relatively short-term (25 weeks) treatment with one of the alkylating agents 1,2,-dimethylhydrazine (DMH) or azoxymethane, we have investigated the mutational spectrum of the ß-catenin gene in a panel of rat colon tumors induced by long-term (20 weeks) DMH-treatment. We detected ß-catenin mutations in 12 of 33 (36%) tumors. Interestingly, only one of the ß-catenin mutations found affected the previously implicated codon 33 cluster region (Asp32Asn), whereas 11 of 12 (>90%) mutations represented identical C
T transitions within codon 41 resulting in the common replacement of threonine by isoleucine. We propose a model in which codon 41 mutations bear higher oncogenic potential but are induced by DMH less frequently than mutations in the codon 33 cluster region. Consequently, only after sustained carcinogenic treatment, as is achieved in the long-term DMH-protocol, codon 41 mutations will be induced frequently enough to be present in all developing malignant lesions and, then, because of their higher oncogenic potential, these are selected for.
Abbreviations: AOM, azoxymethane; APC, human adenomatous polyposis coli gene; ; CTNNB1, human ß-catenin gene; ; Ctnnb1, mouse/rat ß-catenin gene; DMH, 1,2,-dimethylhydrazine
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Introduction
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The ß-catenin protein is a key regulator of the cadherin-mediated cellcell adhesion system by linking the cytoplasmic domain of cadherins to
-catenin, which anchors the adhesion complex to the cytoskeleton (1). ß-Catenin also plays a pivotal role in the Wnt-signal transduction pathway. Activation by Wnt/wingless leads to inhibition of the APCGSK-3ß (glycogen synthase kinase-3ß) complex that, normally, causes phosphorylation of ß-catenins N-terminus and targets its cytoplasmic pool for ubiquitin-mediated degradation (2,3). Upon accumulation, ß-catenin interacts with members of the lymphoid enhancer factor/T-cell factor (Lef-1/TCF) family to generate a functional transcription factor complex (4,5) that causes constitutive transcriptional activation of distinct target genes, e.g. c-myc (6). Stabilization and accumulation of cytoplasmic ß-catenin may result from inactivating mutations in one of its upstream negative regulators, the APC tumor suppressor gene, or from activating mutations in CTNNB1, the gene encoding ß-catenin, itself. ß-Catenin mutations usually occur adjacent to or among the four regulatory phosphorylation sites (codons 33, 37, 41 and 45) located at the N-terminus of the ß-catenin protein. They are causatively associated with a variety of human malignancies including tumors of the skin, brain, kidney and colon (711) and they have been found, especially, in ~50% of human colon tumors possessing an intact APC gene (12).
Colon tumors can be reliably induced in rodents by the repeated administration of the carcinogen 1,2-dimethylhydrazine (DMH) or one of its active metabolites azoxymethane (AOM) (13,14). Irrespective of the mode of administration, these agents specifically induce tumors within the descending colon and which appear histopathologically similar to human sporadic colon tumors. Consequently, chemically induced colon tumors have been widely used to study different aspects of colon carcinogenesis, especially to develop novel therapeutical as well as cancer preventive strategies (15,16).
Recently ß-catenin mutations have been reported to occur at high frequency in AOM-induced rodent colon tumors (17,18). The ß-catenin mutations found were not randomly distributed among the four regulatory GSK-3ß phosphorylation sites but, instead, clustered focally around codon 33. A similar pattern of ß-catenin mutations was observed in a study using a tumor-initiation protocol involving short-term DMH-treatment (once per week; 5 weeks) in rats (19,20). Interestingly, upon a simple post-initiation treatment with the non-carcinogenic phytochemicals chlorophyllin or indole-3-carbinol the characteristic pattern of ß-catenin mutations affecting codon 33 or adjacent residues (80% of all mutations) could be significantly shifted to a pattern where almost half of the mutations directly altered codon 37, 41 or 45. The latter mutations have been proposed to possess higher oncogenic potential. We report here that using a complete carcinogenic protocol in Wistar rats that involves the weekly application of DMH over a time-period of 20 weeks, mutations in Ctnnb1, the rat gene encoding ß-catenin, can be induced that almost exclusively affect codon 41.
To induce colon tumors, male Wistar rats (Charles River, Sultfeld, Germany) at 6 weeks of age were treated with DMH by subcutaneous injection at a dose of 20 mg/kg once weekly. Water and Altromin 1324Nff diet (Altromin, Lage, Germany) were given ad libitum. Animals were killed 1 week after the last injection and colons were removed. Forty-two of 44 treated animals (95%) had colon tumors, the multiplicity (number of tumors per colon) was four (range 111). Tumors were cut out, fixed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin for histological examination. All tumors were histologically classified as adenocarcinomas.
To examine whether DMH-induced rat colon tumors bear mutations in Ctnnb1 total genomic DNA was isolated from sections of 33 paraffin-embedded tumors. Briefly, paraffin was melted by incubating the samples at 95°C for 10 min and then immediately centrifuged at high speed. The paraffin forming a solid lid at the upper phase was scraped off and the remaining samples were digested with proteinase K (Sigma, Taufkirchen, Germany) for 12 h at 56°C. Exon 3 of Ctnnb1, encoding the regulatory degradation targeting box of ß-catenin, was amplified by PCR using primers RNbcat-fwd1 (5'-GCTGACCTGATGGAGTTGGA-3') and RNbcat-rev1 (5'-GCTACTTGCTCTTGCGTGAA-3'). Amplification was performed for 35 cycles in a reaction containing 2 mM MgCl2 and 1 U Platinum-Taq Polymerase (Life Technologies, Rockville, MD, USA). Cycling conditions were: initial denaturation for 5 min at 94°C, 35 cycles of denaturation for 30 s at 94°C, 30 s annealing at 55°C and 30 s elongation at 72°C, followed by a final elongation step for 7 min at 72°C. The resulting PCR fragments were purified using High Pure PCR Purification kit (Roche Diagnostics, Basel, Switzerland) essentially as recommended by the manufacturer, and subsequently subjected to cloning or direct sequencing. For cloning, purified PCR fragments were ligated into the vector pCR 2.1 using the TA cloning system (Invitrogen, Renfrewshire, Scotland, UK) and plasmid DNA was purified by using High Pure Plasmid Isolation kit (Roche Diagnostics, Basel, Switzerland).
Sequencing reactions were set up using either 30 ng of purified PCR fragment or 250 ng of plasmid DNA as template and 10 pmol of sequencing primer in a total reaction volume of 10 µl following a dye terminator protocol (Big Dye, Perkin Elmer, Foster City, CA, USA). The sequencing reactions were run on an ABI Prism 310 DNA Sequencer (Perkin Elmer). Sequencing primers of Ctnnb1 exon 3 were RNbcat-fwd1 and RNbcat-rev2 (5'-CTTGCTCCCACTCATAAAGG-3').
By screening 33 specimens for mutations in Ctnnb1, we detected 12 (36%) samples that displayed sequence ambiguities in their primary PCR products, indicating the presence of non-identical alleles. Upon cloning, we identified two different types of single nucleotide changes that have occurred in these tumors (Table I
). Of the 12 mutations found, 11 were identical point mutations of codon 41 (ACC
ATC) that result in the replacement of a functionally important threonine phosphorylation site by isoleucine, one tumor displayed a mutation of codon 32 (GAT
AAT) that causes a change from aspartate to asparagine. We verified the identity of the mutations by sequencing both of the DNA strands and by analyzing a second, independently generated PCR amplicon. All mutations found were G:C
A:T transitions and were present in a heterozygous state in the tumor whereas no mutations were found in the DNA when extracted from normal colon mucosa of the same animal (not shown). A representative example of the mutational analysis of Ctnnb1 illustrating the predominant codon 41 mutation is shown in Figure 1A
.

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Fig. 1. Nucleotide sequences of the predominant ß-catenin and k-ras mutations in DMH-induced rat colon tumors. Electropherograms for the regions comprising codon 41 of Ctnnb11 (A) and codon 12 of the ki-ras gene (B) showing wild-type sequences (wt) derived from control samples (upper panel), sequences derived from direct sequencing of tumor-derived (tu) PCR-fragments (middle panel), and sequences of isolated mutant alleles as obtained after subcloning (lower panel). Affected codons are boxed, affected nucleotides are indicated by an arrow.
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Using the same DNA samples employed for mutational analysis of Ctnnb1, we screened exon 1 of the ki-ras gene for specific alterations by PCR-sequencing. Exon 1 of ki-ras was amplified using primers RNkras-fwd1 (5'-CCTGCTGAAAATGACTGAGTA-3') and RNkras-rev1 (5'-TTTCAAACAAAGGATGACTG-3'). Cycling conditions were: initial denaturation for 5 min at 94°C, 35 cycles of denaturation for 30 s at 94°C, 30 s annealing at 52°C, and 30 s elongation at 72°C, followed by a final elongation step for 7 min at 72°C.
Sequencing primers of ki-ras exon 1 were RNkras-fwd1 and RNkras-rev2 (5'-CTATCGTAGGATCATATTCA-3'). Twenty-one of 33 (64%) tumors were found to harbor k-ras mutations, 19 of which were G
A transitions at the second base of codon 12 and the remaining two were G
A transitions at the second base of codon 13 (Table I
). A representative example of the mutational analysis of ki-ras illustrating the predominant codon 12 mutation is shown in Figure 1B
.
To test for associations between mutations in Ctnnb1 and ki-ras, we calculated Cohen's Kappa coefficient together with the corresponding 95% confidence interval. We found no statistically significant association of Ctnnb1 and ki-ras mutations (
=0.07; 95% confidence interval ranging from 0.37 to 0.23). The exact two-sided P-value for testing if agreement is purely random was 0.72 which does not allow to conclude more than random agreement.
Mutations in the degradation targeting box should lead to intracellular accumulation and nuclear translocation of ß-catenin protein. In accordance with this, immunohistochemical staining specific for ß-catenin revealed increased cytoplasmic levels of ß-catenin protein and scattered nuclear positive cells in tumor tissues as compared with normal colon tissue. Interestingly, nuclear ß-catenin immunoreactivity was not only restricted to tumors harboring a mutant ß-catenin allele but was also readily detectable in the majority of tumors bearing wild-type ß-catenin (data not shown).
In this study, we have analyzed the status of the Ctnnb1 and ki-ras proto-oncogenes in a panel of 33 DMH-induced rat colon tumors. We detected 12 of 33 (36%) ß-catenin mutations and 21 of 33 (64%) ki-ras mutations. We found that ß-catenin mutations occur either in the presence or in the absence of ki-ras mutations and there was no statistically predilection for either situation. These findings strongly suggest that both the wnt-signaling pathway and the ki-ras pathway act independently of each other and that mutations affecting both pathways contribute in a synergistic manner to rat colon carcinogenesis.
Interestingly, the mutations in Ctnnb1 almost exclusively affected codon 41 and resulted in a single amino acid substitution replacing threonine by isoleucine. At the nucleotide level, this specific mutation corresponds to a G:C
A:T transition, a type of mutation which is recognized to be the preferential mutational event caused by DMH. The finding of a specific mutational hot-spot within codon 41, at a first glance, contrasts with the results of two recent studies that have reported on the preferential mutation of residues flanking codon 33, instead, in either AOM- or DMH-induced rat colon tumors (18,20; see also Figure 2
). Whereas those previous studies, however, used an intiation protocol of rat colon carcinogenesis that involved short-term (25 weeks) carcinogenic treatment and long-term (3652 weeks) follow-up, our study has employed a complete DMH-protocol that involved sustained carcinogenic treatment with DMH for a period of 20 weeks and results in the immediate formation of colon carcinomas in 95% of treated animals at a relative high multiplicity of four tumors per animal. Although, Wistar rats fed Altromin diet have been used in the present study and F344 rats fed AIN-93G diet have been used in at least one of the previous studies (20), the different rat strains and diets are unlikely to be responsible for the different ß-catenin mutations observed, because it has been shown that mice treated with AOM and fed AIN-76A diet display virtually an identical mutational spectrum in Ctnnb1 (hot-spot mutations clustering around codon 33) as rats treated with DMH and fed AIN-93G (17,20). Therefore, it seems more likely that the different mutational spectra observed in Ctnnb1 directly relate to the particular carcinogenic treatment. We propose the following model to explain those different findings (Figure 3
).

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Fig. 2. Distribution of ß-catenin mutations among chemically induced rat colon tumors after treatment with (A) azoxymethane (15 mg/kg) once a week for 2 weeks and followed-up for 36 weeks (18) (B) 1,2-dimethylhydrazine (20 mg/kg) once a week for 5 weeks and followed-up for 52 weeks (20) (C) 1,2-dimethylhydrazine (20 mg/kg) once a week for 5 weeks followed by continuous application of 0.001% chlorophyllin for 52 weeks (20) (D) 1,2-dimethylhydrazine (20 mg/kg) once a week for 20 weeks, no follow-up (this study). The wild-type ß-catenin sequence is shown encompassing the four regulatory glycogen synthase kinase-3ß phosphorylation sites highlighted in bold. The amino acid substitutions reported in the different studies are given above each line in bold.
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Fig. 3. Hypothetical model for the induction of different ß-catenin mutational spectra by DMH. In the initiation protocol (shown left), short-term treatment with DMH induces significantly more mutations affecting the codon 32/33/34 cluster region (x) than codon 41 (o). Tumor outgrowth, however, only occurs beyond a critical threshold level of mutations in cells that have spontaneously acquired additional genetic lesions. In the complete protocol (shown right) basically the same mutational spectrum is induced. However, as a result of sustained carcinogenic treatment cells harboring all different kinds of Ctnnb1 mutations reach the threshold level almost simultaneously. In this situation, competition between neighboring cell clones plays an important role and mutations that confer higher oncogenic activity may expand at the expense of weaker oncogenic mutations.
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In the initiation protocol, short-term treatment with DMH induces significantly more mutations within the codon 32/34 cluster region than mutations in codon 41 and this spectrum simply reflects the intrinsic mutagenic capacity of DMH (or AOM) which has frequently been found to cause mutations specifically in CTGGA motifs that are also present in codon 32 and 34 of Ctnnb1 (18,2022). However, in the short-term protocol, additional genetic lesions have to be acquired by the affected cells in order to reach the critical threshold level of mutations necessary to allow autonomous tumor outgrowth. Only mutations induced frequently (e.g. codons 32 and 34), under these circumstances, have a high probability to show up in tumors whereas the rare mutations (e.g. codon 41) are lost.
Using the complete protocol, virtually the same mutational spectrum is induced, simply because DMH does cause these mutations, specifically. However, as a result of sustained carcinogenic treatment, which is also evidenced by a high frequency and multiplicity of tumor induction, there are so many additional genetic defects induced simultaneously in other oncogenes and tumor suppressor genes that cells harboring all different kinds of Ctnnb1 mutations immediately reach the threshold level. In this situation, simultaneous tumor outgrowth occurs and competition between neighboring cell clones is a critical issue, in which the `stronger' mutations, those which have an even higher oncogenic potential, may take over. According to this model, mutations of codon 41 would be induced at relatively low frequency by DMH for mechanistical reasons, but because of their higher oncogenic activity these mutations still possess the capacity to become the predominant genetic lesion as the mutational system becomes more and more saturated.
In agreement with this hypothetical model, Blum and colleagues (20) reported a significant shift of the Ctnnb1 mutational spectrum when they applied a low-dose post-initiation treatment with chlorophyllin (0.001%) on top of a short-term DMH-protocol (Figure 2C
). Regarded as a chemopreventive agent, chlorophyllin, in these studies, turned out to be a tumor promoter, instead it not only increased the multiplicity of colon tumors significantly (19) but also shifted the Ctnnb1 mutational spectrum from codons 32/34 to codons 41/45 (20). Furthermore, the authors found significantly higher levels of c-jun protein, a putative ß-catenin target gene, in at least two tumors containing a codon 41 mutation as compared with three tumors containing mutations in codon 32 or 34 providing further evidence for the idea that the biological activity of mutated ß-catenin varies depending on the exact type of mutation. Final proof, however, of the differential biological activities exerted by individual ß-catenin mutations will require extensive follow-up studies and these should involve measuring different levels of in vitro and in vivo transforming activity as well as determining the specific transactivation capacity of the various mutant ß-catenin isoforms.
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Notes
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2 To whom correspondence should be addressed Email: r.koesters{at}dkfz.de 
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Acknowledgments
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We thank S.Winkler, I.Voehringer and N.Decker for excellent technical assistance. This work was supported by the Tumorzentrum Heidelberg/Mannheim, Germany.
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Received June 15, 2001;
revised August 10, 2001;
accepted August 24, 2001.