Mutational analysis of Ctnnb1 and Apc in tumors from rats given 1,2-dimethylhydrazine or 2-amino-3-methylimidazo[4,5-f]quinoline: mutational 'hotspots' and the relative expression of beta-catenin and c-jun

Abstract

There is growing interest in beta-catenin and its role in various human cancers. We recently reported that 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)- and 1,2-dimethylhydrazine (DMH)-induced colon tumors in the rat contain mutations in Ctnnb1, the gene for beta-catenin, but the mutation spectrum was influenced by postinitiation exposure to chlorophyllin (CHL) and indole-3-carbinol (I3C) [Blum et al., Carcinogenesis 2001;22:315-320]. The present paper describes a follow-up study in which all of the target organs for IQ- and DMH-induced tumorigenesis were screened; Ctnnb1 mutations were found in 44 of 119 DMH-induced colon tumors, six of 13 IQ-induced colon tumors, 28 of 81 DMH-induced small intestine tumors, none of five IQ-induced small intestine tumors, four of 106 IQ-induced liver tumors, none of 14 DMH-induced Zymbal's gland tumors, none of 24 IQ-induced Zymbal's gland tumors, and none of 29 IQ-induced skin tumors. In tumors from rats given carcinogen alone, or carcinogen plus CHL or I3C, Ctnnb1 mutations frequently substituted amino acids adjacent to Ser33, a critical Ser/Thr residue in the glycogen synthase kinase-3beta regulatory domain of beta-catenin. However, substitution of critical Ser/Thr residues themselves was detected in only three of 24 (12.5%) of the tumors from rats given carcinogen alone, compared with 23 of 58 (40%) of the tumors from rats given carcinogen and treated postinitiation with I3C or CHL (P < 0.02). More than 50 of the colon tumors with wild-type beta-catenin were examined further for their Apc status; the overall frequency of Apc mutations was <10%, and these genetic changes occurred exclusively in the 'Mutation Cluster Region' of Apc. A subset of colon tumors also was examined for expression of beta-catenin and c-jun; these proteins were overexpressed in all tumors containing Ctnnb1 mutations, but the expression was highest in tumors with Ctnnb1 mutations affecting Thr41 and Ser45 residues in the glycogen synthase kinase-3beta region of beta-catenin. Thus, Ctnnb1 mutations occurred more frequently than Apc mutations in colon and small intestine tumors of the rat, and certain mutations upregulated beta-catenin/T-cell factor target genes more effectively than others, perhaps influencing the response to phytochemicals administered postinitiation.

Figure 1

Single-strand conformation polymorphism (SSCP) screening and sequencing of exon 2 of rat Ctnnb1, the region that corresponds to exon 3 of human CTNNB1, and codes for the GSK-3β domain of β-catenin [22]. (a) Ethidium bromide–stained agarose gel and (b) SSCP gel, showing two IQ-induced liver tumors that were wild-type (wt) and a third with a deletion (middle lane, Δ^L); (c) sequencing of Δ^L with the reverse primer revealed an in-frame deletion involving codons 32 to 49; (d) representative SSCP patterns for the more common mutations in IQ- and DMH-induced tumors; bands shown with arrows were removed and sequenced. For examples of typical sequencing results, see [16].

Figure 2

Amino acid substitutions in β-catenin. The wild-type (wt) sequence for part of the GSK-3β region of β-catenin is shown in the horizontal gray box, including critical Ser/Thr phosphorylation sites (Ser33, Ser37, Thr41, Ser45). Results from groups given carcinogen alone (DMH or IQ) are shown above the horizontal box, whereas those from groups given carcinogen plus CHL or I3C are shown below. Superscripts indicate the target organ: C, colon; SI, small intestine; L, liver. Δ^L indicates a liver tumor with an in-frame deletion starting in codon 32 (see Figure 1c). Vertical boxes highlight amino acid substitutions in β-catenin that are rarely seen in colon tumors after carcinogen treatment alone. For more complete information on the mutations and the treatment groups, see Table 1. Modified from a figure (containing the colon tumor data alone) published previously (Figure 2 of [16]).

Figure 3

Expression in rat colon tumors of β-catenin (filled bars) and c-jun (hatched bars). Total tissue lysates were prepared from three separate colon tumors for each of the codons mutated in Ctnnb1, and these were subjected to SDS-polyacrylamide gel electrophoresis and probed with antibodies to β-catenin and c-Jun proteins, as reported before [16]. Inset, representative blots from three tumors containing wild-type (wt) β-catenin, three tumors with mutations in codon 41 of Ctnnb1, and three tumors with mutations in codon 32 of Ctnnb1. Normal colonic mucosa and positive controls for the proteins of interest were included in each gel (not shown). Densitometry measurements were obtained with a gel documentation system and associated software; results were first normalized to expression levels of glyceraldehyde-3-phosphate dehydrogenase and then expressed relative to the protein levels in tumors with wild-type β-catenin (assigned an arbitrary expression value of 1.0). Data represent mean ± SD, n = 3 (except for codon 33, which was from a single tumor). The horizontal line is a reference guide indicating the relative expression of 1.0 for c-jun and β-catenin proteins in tumors with wt β-catenin; all of the bars above this line were significantly different from wt controls, and the bars labeled 41 and 45 were significantly different from all others (P < 0.05).