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

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Abstract

Tumors induced in the rat by 1,2-dimethylhydrazine (DMH) contain mutations in β-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 β-catenin in more than 50 DMH-induced colon tumors and small intestine tumors, and compared this with the concomitant expression of β-catenin mRNA using quantitative real-time RT-PCR analysis. In total, 19/57 (33%) of the tumors harbored mutations in β-catenin, and 14/19 (74%) of the genetic changes substituted amino acids adjacent to Ser33, a key site for phosphorylation and β-catenin degradation. These tumors were found to express a 10-fold range of β-catenin mRNA levels, independent of the β-catenin mutation status and phytochemical exposure, i.e. CHL or I3C given post-initiation. However, β-catenin mRNA levels were strongly correlated with mRNA levels of c-myc, c-jun and cyclin D1, which are targets of β-catenin/Tcf signaling. Tumors with the highest levels of β-catenin mRNA often had over-expressed β-catenin protein, and those with lower β-catenin mRNA typically had low β-catenin protein expression, but there were exceptions (high β-catenin mRNA/low β-catenin protein, or vice versa). We conclude that DMH-induced mutations stabilize β-catenin protein in tumors, which increase c-myc, c-jun and cyclin D1, but there also can be over-expression of β-catenin itself at the mRNA level, contributing to high β-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 β-catenin at the transcriptional level in human and rodent colon cancers.

Introduction

There is growing interest in the β-catenin/T-cell factor (TCF)/lymphoid enhancer factor (LEF) signaling pathway and its role in human cancer development [1]. β-Catenin is a cadherin-binding protein that also functions as a transcriptional activator when complexed in the nucleus with members of the TCF/LEF family [2]. Cytosolic β-catenin interacts with APC, Axin, glycogen synthase kinase-3β (GSK-3β) and other protein partners, leading to phosphorylation of Ser33, Ser37, Thr41 and Ser45 residues in the N-terminal region of β-catenin, followed by ubiquitination and proteosomal degradation [1], [2], [3]. In primary human colon tumors and colorectal cancer cell lines, mutations in CTNNB1 substitute one of the four critical Ser/Thr residues and stabilize β-catenin, leading to accumulation of β-catenin/TCF complexes in the nucleus, and activation of downstream target genes [4], [5], [6].

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. 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].

To complicate matters, the spectrum of β-catenin mutations can be influenced by exposure to dietary phytochemicals, such as chlorophyllin (CHL) and indole-3-carbinol (I3C). 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. High levels of β-catenin and c-Jun were detected in tumors that contained mutations affecting Ser45 or Thr41 of β-catenin, tumors with genetic changes substituting Gly34 and Asp32 had intermediate levels of β-catenin and c-Jun, and the lowest levels of β-catenin and c-Jun were observed in tumors with wild type β-catenin [11].

In the latter experiments, it was assumed that each specific mutation in β-catenin affected its relative stability and turnover at the protein level, the degree of nuclear trafficking and interaction with Tcf/Lef transcription factors, and thus the extent to which downstream target genes became activated. However, in the present investigation of more than 50 DMH-induced tumors in the rat, we observed that β-catenin frequently was over-expressed at the mRNA level. This suggested transcriptional dysregulation of β-catenin, as distinct from the β-catenin protein stabilization reported before [11]. Expression of β-catenin mRNA was correlated with cyclin D1, c-myc and c-jun mRNA levels, but not with the mutation status of β-catenin, or treatment with I3C or CHL.

Section snippets

Source of tumors

Tumors were from a study in which male F344 rats were initiated during the first 5 weeks with DMH (20 mg/kg body weight, by subcutaneous injection), and treated post-initiation with 0.1, 0.01 or 0.001% CHL in the drinking water, or with 0.1, 0.01 or 0.001% I3C in the diet. Full details were provided in the original report [19].

Mutation screening

A subset of 57 DMH-induced small intestine and colon tumors, from each of the treatment groups in the original study, was screened for mutations in β-catenin. The

Confirmation of a mutational ‘hotspot’ involving codons 32 and 34

In the present investigation, 16 tumors from the DMH control group, 25 tumors from the DMH + CHL group, and 22 tumors from the DMH + I3C group were screened using PCR-SSCP analysis (Fig. 1). One of the samples lacked any of the bands for the wild type, indicating that the tumor was likely to be homozygous for the corresponding β-catenin mutation. Sequencing of this tumor identified a mutation in codon 32 of β-catenin (GAT  AAT, D32N). Table 2 shows the complete list of β-catenin mutations, confirmed

Discussion

The present investigation builds upon prior work showing that phytochemicals such as CHL and I3C can alter the spectrum of β-catenin mutations in DMH- and IQ-induced tumors [9], [11]. Under conditions of tumor promotion, there was an increase in mutations affecting Ser37, Thr41 and Ser45 of β-catenin [11]. The latter mutations have been reported in human cancers, but they occur less frequently in the colon tumors from rats exposed to chemical carcinogens in the absence of a tumor promoter [7],

Acknowledgments

Thanks are extended to Meirong Xu, who was responsible for overseeing the original carcinogenicity bioassay. We also thank Qingjie Li for help with primer design, and members of the Tanguay and Hagen laboratories for access to qPCR equipment. Sequencing was performed in the Center for Gene Research and Biotechnology at Oregon State University. This work was supported in part by NIH grants CA65525, CA80176, and CA90890.

References (25)

  • B. Mann et al.

    Target genes of β-catenin-T-cell factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas

    Proc. Natl. Acad. Sci. U.S.A.

    (1999)
  • O. Tetsu et al.

    β-Catenin regulates expression of cyclin D1 in colon carcinoma cells

    Nature

    (1999)
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