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

Abstract

Tumors induced in the rat by 1,2-dimethylhydrazine (DMH) contain mutations in beta-catenin, but the spectrum of such mutations can be influenced by phytochemicals such as chlorophyllin (CHL) and indole-3-carbinol (I3C). In the present study, we determined the mutation status of beta-catenin in more than 50 DMH-induced colon tumors and small intestine tumors, and compared this with the concomitant expression of beta-catenin mRNA using quantitative real-time RT-PCR analysis. In total, 19/57 (33%) of the tumors harbored mutations in beta-catenin, and 14/19 (74%) of the genetic changes substituted amino acids adjacent to Ser33, a key site for phosphorylation and beta-catenin degradation. These tumors were found to express a 10-fold range of beta-catenin mRNA levels, independent of the beta-catenin mutation status and phytochemical exposure, i.e. CHL or I3C given post-initiation. However, beta-catenin mRNA levels were strongly correlated with mRNA levels of c-myc, c-jun and cyclin D1, which are targets of beta-catenin/Tcf signaling. Tumors with the highest levels of beta-catenin mRNA often had over-expressed beta-catenin protein, and those with lower beta-catenin mRNA typically had low beta-catenin protein expression, but there were exceptions (high beta-catenin mRNA/low beta-catenin protein, or vice versa). We conclude that DMH-induced mutations stabilize beta-catenin protein in tumors, which increase c-myc, c-jun and cyclin D1, but there also can be over-expression of beta-catenin itself at the mRNA level, contributing to high beta-catenin protein levels. Similar findings have been reported in primary human colon cancers and their liver metastases, compared with matched normal-looking tissue. Thus, further studies are warranted on the mechanisms that upregulate beta-catenin at the transcriptional level in human and rodent colon cancers.

Fig. 1

DMH-induced tumors in the rat were screened for β-catenin mutations using PCR-based single strand conformation polymorphism (PCR-SSCP) analysis. Each lane marked with an arrow had an altered band pattern compared with wild type (WT) β-catenin, and sequence analysis confirmed the mutation in β-catenin as affecting codon: (a) 32, (b) 33, (c) 34, (d) 37, (e) 41 or (f) 45. One sample (asterisk, *) lacked any of the bands from the corresponding WT control, and was homozygous for D32N mutant β-catenin (see Table 2 for mutations confirmed by sequencing).

Fig. 2

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 [–11], substituting amino acids adjacent to Ser33, which is a critical target for phosphorylation and β-catenin protein degradation.

Fig. 3

β-Catenin mRNA expression varies markedly in DMH-induced tumors. Each tumor was screened by qPCR, and β-catenin mRNA expression was normalized to the corresponding levels for GAPDH. Data bars (mean ± S.E., n = 3) were arranged from lowest to highest mRNA expression for small intestine tumors (black bars) and colon tumors (gray bars), and according to whether β-catenin was wild type (upper panel, n = 35) or mutant (lower panel, n = 19). The lower panel also shows each amino acid substitution identified in β-catenin (e.g. D32N).

Fig. 4

β-Catenin mRNA expression was correlated with cyclin D1, c-myc and c-jun mRNA levels in DMH-induced tumors. qPCR was used to examine DMH-induced tumors and normal mucosa for expression of three reported β-catenin/Tcf downstream targets, as well as AP-2α, a negative regulator of β-catenin/Wnt signaling [20]. In each case, mRNA expression was first normalized to the housekeeping gene, GAPDH. Open symbols (○), tumors containing WT β-catenin; closed symbols (●), tumors with mutant β-catenin.

Fig. 5

β-Catenin mRNA expression in DMH-induced tumors was unrelated to chlorophyllin (CHL) or indole-3-carbinol (I3C) treatment. qPCR was used to determine β-catenin mRNA expression relative to GADPH, as described in Fig. 4. Data = mean ± S.E., for the number of tumors (n) shown above each bar, and for the number of normal mucosal samples from control rats given no DMH. Tumors analyzed here were from a larger study [19] in which rats were treated with DMH and then exposed post-initiation to one of three different concentrations of CHL in the drinking water, or I3C in the diet, as indicated in the figure.

Fig. 6

Concordance between β-catenin mRNA and protein levels in DMH-induced tumors. Immunoblotting was performed for β-catenin, with β-actin as loading control, and protein expression was compared with the corresponding levels of β-catenin mRNA. Values shown above each lane were obtained from Fig. 3, and represent β-catenin mRNA levels normalized to GAPDH. ND, not detected.