-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

Abstract

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 beta-catenin, but the pattern of mutation differs from that found in human colon cancers. In both species, mutations affect the glycogen synthase kinase-3beta consensus region of beta-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 beta-catenin mutations in rat colon tumors induced by IQ and DMH. Specifically, 17/39 (44%) of the beta-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 beta-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 beta-catenin. The results indicate that the pattern of beta-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 beta-catenin/Tcf/Lef target genes.

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

“…The methodology for PCR-based single strand conformation polymorphism (PCR-SSCP) analysis coupled with direct sequencing was reported elsewhere [9].…”

“…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.…”

“…First, as in the human situation, colon tumors in the rat contain mutations in Apc or Ctnnb1, but not in both of these genes [7]. Second, β-catenin/Tcf downstream targets frequently are over-expressed, including c-Myc, c-Jun and cyclin D1 [9,11]. Third, genetic changes in human CTNNB1 or murine Ctnnb1 substitute amino acids within the GSK-3β regulatory domain of β-catenin.…”

“…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].…”

“…The latter compound is found in cruciferous vegetables [12][13][14], whereas CHL is a water soluble derivative of chlorophyll, the ubiquitous pigment in green, leafy vegetables [15][16][17]. In the tumors from rats given DMH or IQ alone, virtually all of the β-catenin mutations substituted amino acid residues adjacent to Ser33, whereas in animals given carcinogen followed by I3C or CHL, β-catenin mutations more often substituted one of the critical Ser/Thr residues [9]. Subsequent work [18] showed that amino acid substitutions adjacent to Ser33 retard, rather than completely block, the proteasome degradation pathway, leading to a range of β-catenin protein expression levels in colon tumors.…”