ß-Catenin mutation in rat colon tumors initiated by 1,2-dimethylhydrazine and 2-amino-3-methylimidazo[4,5-f]quinoline, and the effect of post-initiation treatment with chlorophyllin and indole-3-carbinol

Carmen A. Blum1,2, Meirong Xu1, Gayle A. Orner1,2, Arthur T. Fong1, George S. Bailey2, Gary D. Stoner3, David T. Horio4 and Roderick H. Dashwood1,2,5

1 Linus Pauling Institute and
2 Department of Environment and Molecular Toxicology, Oregon State University, Corvallis, OR 97331-6512,
3 School of Public Health, Ohio State University, Columbus, OH 43210 and
4 Department of Pathology, St Francis Medical Center, Honolulu, HI 96817, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carcinogens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 1,2-dimethylhydrazine (DMH) induce colon tumors in the rat that contain mutations in ß-catenin, but the pattern of mutation differs from that found in human colon cancers. In both species, mutations affect the glycogen synthase kinase-3ß consensus region of ß-catenin, but whereas they directly substitute critical Ser/Thr phosphorylation sites in human colon cancers, the majority of mutations cluster around Ser33 in the rat tumors. Two dietary phytochemicals, chlorophyllin and indole-3-carbinol, given post-initiation, shifted the pattern of ß-catenin mutations in rat colon tumors induced by IQ and DMH. Specifically, 17/39 (44%) of the ß-catenin mutations in groups given carcinogen plus modulator were in codons 37, 41 and 45, and substituted critical Ser/Thr residues directly, as seen in human colon cancers. None of the tumors from groups given carcinogen alone had mutations in these codons. Interestingly, many of the mutations that substituted critical Ser/Thr residues in ß-catenin were from a single group given DMH and 0.001% chlorophyllin, in which a statistically significant increase in colon tumor multiplicity was observed compared with the group given DMH only. These tumors had marked over-expression of cyclin D1, c-myc and c-jun mRNA and c-Myc and c-Jun proteins were strongly elevated compared with tumors containing wild-type ß-catenin. The results indicate that the pattern of ß-catenin mutations in rat colon tumors can be influenced by exposure to dietary phytochemicals administered post-initiation, and that the mechanism might involve the altered expression of ß-catenin/Tcf/Lef target genes.

Abbreviations: APC, human adenomatous polyposis coli gene; Apc, rat adenomatous polyposis coli gene; CHL, chlorophyllin; CTNNB1, human ß-catenin gene; Ctnnb1, rat ß-catenin gene; DMH, 1,2-dimethylhydrazine; GSK-3ß, glycogen synthase kinase-3ß; I3C, indole-3-carbinol; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; LEF, lymphoid enhancer factor; PCR, polymerase chain reaction; PhlP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; SSCP, single strand conformation polymorphism; TCF, T-cell factor.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Colorectal cancer is a leading cause of cancer-related deaths in the US (1). Cancers of the colon and rectum are believed to arise via mutations in oncogenes, tumor suppressor genes and genes that control DNA repair and replication. Among these genes, APC has been described as the `gatekeeper' of colorectal cancer because it is mutated in 80–85% of human colorectal cancers (2). However, recent studies have shown that human colon tumors with wild-type APC contain mutations in CTNNB1 (35). The product of this gene, ß-catenin, is a cadherin-binding protein involved in cell–cell adhesion (6), but also functions as a transcriptional activator when complexed in the nucleus with members of the TCF/LEF family of binding proteins (7,8). Control of the cytosolic levels of ß-catenin is through the Wnt signal transduction cascade, in which the protein complex consisting of APC, axin and the serine kinase glycogen synthase kinase-3ß (GSK-3ß), negatively regulate ß-catenin (9,10). In the absence of Wnt signal, GSK-3ß phosphorylates ß-catenin at critical Ser/Thr residues, targeting it for ubiquitination by ß-TrCP and proteosomal degradation (11,12). In primary human colon tumors and colorectal cell lines, mutations in CTNNB1 substitute the critical Ser/Thr residues in the GSK-3ß region and stabilize ß-catenin, leading to the accumulation of ß-catenin–TCF–LEF complexes in the nucleus (35). This complex is able to activate various target genes, including c-MYC, c-JUN and cyclin D1 (1315).

In addition to genetic factors, a major risk factor for colorectal cancer is diet, and approximately one third of human colorectal cancers might be prevented through appropriate dietary modification (16). The human diet contains mutagens and carcinogens, as well as cancer chemopreventive compounds (1720). Two such compounds that have been of interest in this laboratory are indole-3-carbinol (I3C), a constituent of brassica vegetables, and chlorophyllin (CHL), a water-soluble salt of chlorophyll. Previous studies have determined that during the initiation phase, I3C and CHL exhibit anti-carcinogenic properties by altering carcinogen metabolizing enzymes or via molecular complex formation, respectively (21,22). However, the post-initiation effects of these compounds are generally less well characterized, and a few studies have provided evidence for tumor promotion or enhancement depending upon the initiator, exposure protocol and species (see refs 21 and 22 for mini-reviews).

In this report, we examined the pattern of ß-catenin mutations in colon tumors from rats induced with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 1,2-dimethylhydrazine (DMH) and post-treated with CHL or I3C. The carcinogen DMH is a synthetic compound that induces tumors of the colon and small intestine in the rat (23), whereas IQ is generated during normal cooking of meat and fish (24), and produces tumors of the liver, colon, small intestine, Zymbal's gland and skin (25). Colon tumors induced by DMH contain Ki-ras mutations but lack genetic changes in p53 (26), whereas those induced by IQ lack mutations in Ki-ras and p53 genes, and have few microsatellite or Apc mutations (27). However, recent studies have established that the tumors induced by IQ and other colon carcinogens in the rat contain mutations in Ctnnb1 (2830). We report here that the pattern of ß-catenin mutations in carcinogen-induced rat colon tumors can be altered by post-initiation treatment with CHL or I3C.


    Materials and methods
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 Materials and methods
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 References
 
Source of colon tumors
Colon tumors were from a 1 year study in which male F344 rats were initiated with DMH or IQ during the first 5 weeks of the experiment, and 1 week later the animals were treated with CHL or I3C until termination. For information on doses and routes of administration, see Table IGo. At the time of necropsy, in addition to taking tissue for histological examination (31), a portion of each tumor was frozen on liquid nitrogen and stored at –80°C for molecular analyses.


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Table I. Summary of ß-catenin mutations in DMH and IQ groups (rat colon tumorsa)
 
Mutation screening and sequencing
Tissue samples were extracted with DNAzol genomic isolation reagent (Molecular Research Center), and the DNA was quantified using a Shimadzu UV-2401PC spectrophotometer. Amplification of the region corresponding with the GSK-3ß region of ß-catenin was carried out using the polymerase chain reaction (PCR), according to the conditions described before (28). Single-strand conformation polymorphism (SSCP) screening was performed using the GenePhor electrophoresis system (Amersham Pharmacia Biotech). DNA (6 ng/well) was run at 15°C, 600 V, 25 mA and 15 W for 1.5 h, and the gel was silver stained. Bands exhibiting altered migration were cut from the gel, re-amplified and cleaned using Wizard PCR Preps (Promega). Samples were sequenced in both directions on an ABI PrizmTM model version 3.3 automated sequencer.

RT–PCR
Frozen tumor samples were added to the Micro-FastTrack 2.0 kit (Invitrogen) in order to isolate mRNA, and either Superscript II (Gibco BRL) or the Promega RT system was used to synthesize cDNA. Primer sequences and PCR conditions were as published for hprt and cyclin D1 (32), c-myc (33) and c-jun (34).

Western blotting
Tumor tissue was homogenized at room temperature in RIPA lysis buffer (Qiagen) and centrifuged at 14 000 g for 2 min through the QIAshredder (Qiagen). Protein (15–20 µg) was run on a 4–12% Bis-Tris gel (Novex) and transferred onto a nitrocellulose membrane. After overnight blocking, the membrane was first incubated for 1 h with primary antibody, then washed and incubated with secondary antibody conjugated with horseradish peroxidase (Bio-Rad), and finally developed using ECL reagents 1 and 2 (Amersham). Imaging and quantification of the data was by an Alphalnnotech photodocumentation system. The primary antibodies used were as follows: ß-catenin, mouse monoclonal antibody (Transduction Labs), 1:600 dilution; c-Jun, mouse monoclonal antibody (Transduction Labs), 1:1000 dilution; c-Myc, rabbit polyclonal antibody (Santa Cruz), 1:100 dilution; Cyclin D1, rabbit polyclonal antibody (NeoMarkers), 1:200 dilution.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
DNA from more than 130 colon tumors was successfully screened by PCR–SSCP analysis and the mutations confirmed by sequencing (Figure 1Go; Table IGo). Genetic changes were found in codons 32, 33, 34, 37, 41 and 45 of Ctnnb1, and substituted critical Ser/Thr residues or their adjacent amino acids within the GSK-3ß region of ß-catenin. The majority of these tumors analyzed were from groups given DMH; ß-catenin mutations were detected in 44/119 (37%) of the DMH-induced tumors versus 6/13 (46%) of the tumors from groups given IQ (Table IGo, final column). Due to the low number of IQ-induced tumors, no information was obtained on the effect of I3C or CHL treatment on the spectrum or frequency of ß-catenin mutations.



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Fig. 1. Single strand conformation polymorphism (SSCP) results (A) and sequencing data (B) for ß-catenin mutations in colon tumors induced by DMH or IQ in the F344 rat. `M', marker ladder; lane 1, wild-type pattern; lanes 2–6, tumors containing shifted bands (e.g. arrows), confirmed by sequencing (i)–(v) to involve codons 32, 34, 37, 41 and 45. Mutations also were found in two instances involving codon 33 (not shown).

 
In the DMH groups, no differences were seen in the overall frequency of ß-catenin mutations when the combined groups given CHL or the combined groups given I3C were compared with the positive control group given carcinogen alone. An inverse relationship between concentration of CHL and ß-catenin mutation frequency was observed; thus, 23, 44 and 50% of the tumors had mutations for 0.1, 0.01 and 0.001% CHL groups, respectively, but this trend proved not to be statistically significant when the analysis included the group given DMH alone. In the group given DMH alone, 10/30 (33%) of the colon tumors had ß-catenin mutations, and sequencing revealed that these were clustered in codons 32, 33 and 34; 8/10 (80%) were G->A transition mutations affecting codons 32 and 34 (Table IGo). Codons 32 and 34 also were hotspots for mutation in groups given carcinogen and post-treated with CHL or I3C; however, in marked contrast to the results seen after treatment with carcinogen alone, 17/39 (44%) of the genetic changes directly altered codons 37, 41 or 45.

When the results from this study were summarized in terms of the amino acid substitutions in the ß-catenin protein, a shifted pattern of mutations was clearly apparent in the groups given CHL or I3C; thus, genetic changes affecting Ser37, Thr41 and Ser45 were identified exclusively in the groups given carcinogen and post-treated with either modulator (Figure 2Go, gray boxes). Over half of these mutations (9/17 = 53%) were from groups given DMH or IQ and post-treated with 0.001% CHL. In one group, namely DMH + 0.001% CHL, screening of Ctnnb1 mutations identified substitutions in Ser37, Thr41 or Ser45 residues as the most common type of genetic change (8/10 = 80%). Because this was the group in which a statistically significant increase in colon tumor multiplicity was observed compared with animals given DMH alone (31), we next examined the expression of possible ß-catenin/Tcf/Lef target genes, comparing tumors with wild-type versus mutant forms of ß-catenin.



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Fig. 2. Summary of amino acid substitutions in the ß-catenin protein. The wild-type sequence corresponding with part of the glycogen synthase kinase-3ß (GSK-3ß) region of ß-catenin is shown in the horizontal box, with critical phosphorylation sites highlighted with a superscript (Ser33, Ser37, Thr41 and Ser45). Results from groups given carcinogen alone (DMH or IQ) are shown above the horizontal box, whereas those from groups given carcinogen plus modulator are shown below, including shaded (gray) boxes for `unique' substitutions not seen in groups given carcinogen alone. Results for CHL or I3C are shown by the corresponding superscript, and for emphasis those from the groups given 0.001% CHL or 0.001% I3C are underlined (e.g. FCHL or NI3C).

 
The expression of cyclin D1 mRNA was first examined in six colon tumors containing ß-catenin mutations (Figure 3AGo, upper panel); a marked increase in cyclin D1 expression was detected in all tumors compared with the expression of this gene in normal colonic mucosa. Tumors were further examined for changes in expression of c-myc and c-jun. The expression of both genes was readily detected at the mRNA level under conditions in which normal colonic mucosa (`N') produced no obvious product (except for the housekeeping gene, hprt, upper band in each gel).



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Fig. 3. Expression of ß-catenin/Tcf/Lef target genes in rat colon tumors from the group treated with DMH plus 0.001% CHL. (A) RT–PCR analyses of cyclin D1 (top), c-myc (middle) and c-jun relative mRNA expression (bottom) in normal tissue (`N') and in six colon tumors known to contain mutations in ß-catenin. The upper band in each gel is for the house-keeping gene Hprt. (B) RT–PCR analyses of hprt, cyclin D1, c-myc and c-jun relative mRNA expression in five colon tumors containing wild-type ß-catenin. (C) Western blot of ß-catenin, c-Jun, c-Myc and cyclin D1 proteins in rat colon tumors. Total tissue lysate was separated by gel electrophoresis, and detection was by monoclonal antibodies to the proteins of interest (see Materials and methods for details). Lanes 1–5, colon tumors containing wild-type ß-catenin; lanes 6–10, tumors containing mutant ß-catenin; lane 11, normal colonic mucosa (`N'). Protein standards and lysates from various cell lines were used on each gel as positive controls to verify the positions of the proteins of interest (not shown).

 
Five colon tumors with wild-type ß-catenin were next screened by RT–PCR for changes in cyclin D1, c-myc and c-jun (Figure 3BGo). Cyclin D1 was detected in all five tumors but the expression was approximately equivalent to that of Hprt in each case (for contrast, see Figure 3AGo, upper panel). In three of the tumors, c-myc and c-jun mRNA was not detected, and these genes were only marginally expressed in the remaining two tumors.

To determine whether the changes in mRNA levels were paralleled by alterations in protein expression, the same tumors were examined by Western blotting (Figure 3CGo). These studies confirmed that in tumors bearing Ctnnb1 mutations (Figure 3CGo, lanes 6–10), ß-catenin, c-Jun, c-Myc and cyclin D1 proteins were strongly expressed, in marked contrast to normal colonic mucosa (Figure 3CGo, lane 11). In addition, two of the tumors that were apparently wild-type for Ctnnb1 also showed increased levels of ß-catenin protein (Figure 3CGo, lanes 1 and 2); mutations in other genes, such as Apc, have yet to be examined. None of the tumors with wild-type ß-catenin expressed c-Jun or c-Myc at appreciable levels (Figure 3CGo, lanes 1–5). Cyclin D1 was strongly expressed in all tumors, regardless of ß-catenin status, except lane 3 which had levels comparable with normal colon in lane 11. Scanning densitometry confirmed that, in lanes 6–10, the expression of all four proteins was increased >10-fold compared with normal colonic mucosa from control rats (data not shown).


    Discussion
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 Abstract
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 Materials and methods
 Results
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 References
 
This the first study to demonstrate that the spectrum of ß-catenin mutations in carcinogen-induced rat colon tumors can be altered by post-initiation exposure to phytochemicals. To our knowledge, it is also the first investigation of ß-catenin expression and the expression of three ß-catenin/Tcf/Lef target genes, namely cyclin D1, c-myc and c-jun, in a subset of carcinogen-induced rat colon tumors. A previous study reported on the different frequencies and patterns of ß-catenin mutations in liver tumors induced by N-nitrosodiethylamine or a choline-deficient diet (35). A second report described a decrease in ß-catenin expression in small intestinal and colon polyps of APCmin mice treated with 1,4-phenylene bis(methylene)selenocyanate (36). However, the finding that there is a shift in the distribution of ß-catenin mutations in groups given IQ or DMH and post-treated with CHL or I3C is novel, and has potentially important implications regarding gene–diet interactions and the events that give rise to colon cancers.

In human colorectal cell lines and primary colon cancers, mutations in CTNNB1 usually substitute the critical Ser/Thr residues within the GSK-3ß region of ß-catenin, namely Ser33, Ser37, Thr41 or Ser45 (3,4), and as a result the ß-catenin protein no longer is a substrate for phosphorylation and subsequent degradation by the ubiquitin/proteosome pathway (12,13). In the present study, none of the colon tumors induced by IQ or DMH, in the absence of CHL or I3C treatment, had mutations affecting Ser37, Thr41 or Ser45, and only 2/11 (18%) had mutations that substituted Ser33 (Figure 2Go). Previous studies (2830) of the colon tumors induced by IQ, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP), azoxymethane or methylazoxymethanol acetate plus 1-hydroxyanthraquinone found, collectively, only one mutation that substituted Ser33, two that substituted Ser37, and two that substituted Thr41, whereas the amino acids immediately adjacent to Ser33 were substituted in the majority of tumors with ß-catenin mutations (28/35 = 80%). Most of the ß-catenin mutations in the present study (Table IGo) and elsewhere (2830) involved G->A transitions within two CTGGA sequences (codons 31–34), consistent with the known preference of the carcinogens for forming covalent adducts with guanine bases in DNA (23,26,27).

In this investigation, none of the vehicle controls post-treated with CHL or I3C had tumors, and thus the modulator itself is unlikely to have directly produced the mutations in codons 37, 41 and 45 of Ctnnb1. It is more likely that these mutations occurred as a result of the carcinogen treatment, but the initiated cells did not progress to form colon tumors in the absence of modulator treatment. One interpretation is that I3C or CHL exposure may have provided a survival advantage to the cells containing mutant forms of ß-catenin with substitutions in Ser37, Thr41 or Ser45. Precisely how I3C or CHL might enable survival and clonal expansion of these cells is presently unclear, but the mechanisms most likely involve the genes controlling cell proliferation and apoptosis in the colonic mucosa. We focused on three ß-catenin/Tcf/Lef target genes as candidates for further study. A search of the Genebank showed that the promoter regions of rat cyclin D1, c-myc and c-jun have perfect Tcf/Lef motifs, like their human counterparts (1315), and mRNA levels of all three of these genes were markedly overexpressed in colon tumors containing mutations in Ctnnb1, and there were strongly elevated levels of ß-catenin protein. Interestingly, two of the tumors with codon 41 mutations showed significantly (>3-fold) higher amounts of c-Jun compared with three tumors containing mutations in codons 32 or 34 (Figure 3CGo, compare lanes 6 and 7 with lanes 8–10). The results suggest that some mutant forms of ß-catenin, namely those with direct substitutions in critical Ser/Thr residues, may have a greater ability to upregulate c-Jun than others with amino acid substitutions adjacent to Ser33. These findings in vivo are supported by previous studies (37), in which certain mutant forms of ß-catenin were more effective than others in activating ß-catenin/Tcf/Lef signaling following transient transfection into cell lines.

For the reasons described above, we focused on the expression of candidate ß-catenin/Tcf/Lef target genes in the colon tumors from rats given DMH and 0.001% CHL, but additional studies are now in progress on the colon tumors from other groups in this study. The results are anticipated to expand upon those shown in Figure 3Go by providing, for each specific mutant form of ß-catenin, quantitative data on gene expression relative to an internal standard (competitor or mimic). The hypothesis is that, with some dietary modulators, tumor promotion involves the selection of particular mutant forms of ß-catenin with a more potent ability than others to activate genes controlling key cell proliferation and apoptosis pathways in the rat colon, and possibly human colon. It is interesting to speculate that the spectrum of ß-catenin mutations normally found in human colon cancers might involve initiating events coupled with a selection process, in which dietary constituents enable cells with certain forms of mutant ß-catenin to survive and progress towards tumor formation, when they would otherwise be deleted by apoptosis.


    Notes
 
5 To whom correspondence should be addressed E-mail: Rod.Dashwood{at}orst.edu Back


    Acknowledgments
 
This study was supported in part by NIH grants CA65525, CA80176, CA34732, ES00210 and ES03850. Support for C.A.B. and G.A.O. was provided by NIEHS training grant T32 ES07060.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 18, 2000; revised November 22, 2000; accepted November 27, 2000.