Predominant mutation of codon 41 of the β-catenin proto-oncogene in rat colon tumors induced by 1,2-dimethylhydrazine using a complete carcinogenic protocol

Abstract

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 beta-catenin gene. Mutations found in the beta-catenin gene typically affect one out of four regulatory phosphorylation sites near the N-terminus of the beta-catenin protein. Whereas in human colon cancers, however, the majority of beta-catenin mutations directly alter threonine 41 or serine 45; the beta-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 (2-5 weeks) treatment with one of the alkylating agents 1,2,-dimethylhydrazine (DMH) or azoxymethane, we have investigated the mutational spectrum of the beta-catenin gene in a panel of rat colon tumors induced by long-term (20 weeks) DMH-treatment. We detected beta-catenin mutations in 12 of 33 (36%) tumors. Interestingly, only one of the beta-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.

Tumors from rats given 1,2-dimethylhydrazine plus chlorophyllin or indole-3-carbinol contain transcriptional changes in β-catenin that are independent of β-catenin mutation status

“…In PhIP-treated rats, promotion by a high-fat diet led to an increase in dysplastic aberrant crypt foci and colon tumors, but none of the mutations identified affected critical Ser/Thr residues in β-catenin [21,22]. Conversely, a high frequency of codon 41 β-catenin mutation (Thr41Ile) was found in rats given DMH over a period of 20 weeks as part of a 'complete carcinogen' protocol, without a separate tumor promoting agent [10]. Nonetheless, it is clear from published reports [9][10][11]21,22] that the carcinogen and/or phytochemical exposure protocol can influence the final pattern of β-catenin mutations in colon tumors.…”

“…Conversely, a high frequency of codon 41 β-catenin mutation (Thr41Ile) was found in rats given DMH over a period of 20 weeks as part of a 'complete carcinogen' protocol, without a separate tumor promoting agent [10]. Nonetheless, it is clear from published reports [9][10][11]21,22] that the carcinogen and/or phytochemical exposure protocol can influence the final pattern of β-catenin mutations in colon tumors. Understanding how such patterns arise might provide better insight into the risk factors for human colorectal cancer development.…”

“…Summary of β-catenin mutations in DMH-induced tumors. An apparent mutational 'hotspot' around codons 32 and 34 has been reported in prior studies with colon carcinogens in the rat [7][8][9][10][11], substituting amino acids adjacent to Ser33, which is a critical target for phosphorylation and β-catenin protein degradation. β-Catenin mRNA expression varies markedly in DMH-induced tumors.…”

“…Mutations in β-catenin also have been detected in colon tumors of animals treated with chemical carcinogens, such as 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo [4,5-f] quinoline (IQ), azoxymethane, 1,2-dimethylhydrazine (DMH), and methylazoxymethanol acetate plus 1-hydroxyanthraquinone [7][8][9][10][11]. In fact, there are a number of similarities between human and rat colon tumors with respect to the β-catenin pathway.…”

“…Third, genetic changes in human CTNNB1 or murine Ctnnb1 substitute amino acids within the GSK-3β regulatory domain of β-catenin. However, whereas the vast majority of β-catenin mutations in human colon cancers substitute critical Ser/Thr residues directly, in rat colon tumors the mutations often localize to two CTGGA 'hotspot' sequences and substitute amino acids adjacent to Ser33 [7][8][9][10][11].…”